Methods and compositions for affecting the flavor and aroma profile of consumables

ABSTRACT

This document relates to food products containing highly conjugated heterocyclic rings complexed to an iron ion and one or more flavor precursors, and using such food products to modulate the flavor and/or aroma profile of other foods. The food products described herein can be prepared in various ways and can be formulated to be free of animal products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/913,018 filed Mar. 6, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/398,479 filed Jan. 4, 2017, which is acontinuation of U.S. patent application Ser. No. 14/797,006 filed Jul.10, 2015, which is a continuation of PCT/US2014/011347 filed Jan. 13,2014, which is a continuation of and claims priority to U.S. applicationSer. No. 13/941,211, filed Jul. 12, 2013, U.S. Application Ser. No.61/908,634, filed Nov. 25, 2013, and to U.S. Application Ser. No.61/751,816, filed Jan. 11, 2013, and is related to the followingco-pending patent applications: Application Serial No. PCT/US12/46560;Application Serial No PCT/US12/46552; Application Ser. No. 61,876,676,filed Sep. 11, 2013; and Application Ser. No. 61/751,818, filed Jan. 11,2013, all of which are incorporated herein by reference.

TECHNICAL FIELD

This invention relates to food products and more particularly, to foodproducts that include a highly conjugated heterocyclic ring complexed toiron such as a heme-cofactor and one or more flavor precursor molecules.

BACKGROUND

Food is any substance that is either eaten or drunk by any animal,including humans, for nutrition or pleasure. It is usually of plant oranimal origin, and can contain essential nutrients, such ascarbohydrates, fats, proteins, vitamins, or minerals. The substance isingested by an organism and assimilated by the organism's cells in aneffort to produce energy, maintain life, or stimulate growth.

Food typically has its origin in a photosynthetic organism, such as aplant. Some food is obtained directly from plants, but even animals thatare used as food sources are raised by feeding them food which istypically derived from plants.

In most cases, the plant or animal food source is fractionated into avariety of different portions, depending upon the purpose of the food.Often, certain portions of the plant, such as the seeds or fruits, aremore highly prized by humans than others and these are selected forhuman consumption, while other less desirable portions, such as thestalks of grasses, are typically used for feeding animals.

Current plant-based meat substitutes have largely failed to cause ashift to a vegetarian diet. Meat substitute compositions are typicallyextruded soy/grain mixtures which largely fail to replicate theexperience of cooking and eating meat. Common limitations of plant-basedmeat substitute products are a texture and mouth-feel that are morehomogenous than that of equivalent meat products. Furthermore, as theseproducts must largely be sold pre-cooked, with artificial flavors andaromas pre-incorporated, they fail to replicate the aromas, flavors, andother key features, such as texture and mouth-feel, associated withcooking or cooked meat. As a result, these products appeal largely to alimited consumer base that is already committed tovegetarianism/veganism, but have failed to appeal to the larger consumersegment accustomed to eating meat. It would be useful to have improvedplant-based meat substitutes which better replicate the aromas andflavors of meat, particularly during and/or after cooking.

SUMMARY

Provided herein are methods and compositions for modulating the flavorand/or aroma profile of consumable food products, including animal- ornon-animal (e.g., plant) based food products, or mixtures of animal- andnon-animal-based food products. In some embodiments, the methods andcompositions are useful for modulating the flavor and/or aroma profileof a consumable food product during and/or after the cooking process. Insome embodiments, the methods and compositions are used to generate oneor more chemical compounds that modulate the flavor and/or aroma profileof the consumable food product during and/or after the cooking process.

As provided herein, and without being bound by theory, certaincharacteristic meaty flavors and/or aromas (e.g., beefy, bacony, umami,savory, bloody, brothy, gravy, metallic, bouillon-like; see Tables 2, 7,and 11), including one or more specific chemical compounds associatedwith the same (see Tables 3, 8, 9, 12, 14, 16, or 17), are believed tobe produced during the cooking process of a consumable food product bychemical reaction of one or more flavor precursor molecules orcompositions catalyzed by the presence of a highly conjugatedheterocyclic ring complexed to an iron ion (e.g., a heme moiety; or aporphyrin; a porphyrinogen; a corrin; a corrinoid; a chlorin; abacteriochorophyll; a corphin; a chlorophyllin; a bacteriochlorin; or anisobacteriochlorin moiety complexed to an iron ion). Such highlyconjugated heterocycylic moieties include heterocyclic aromatic ringscomposed of one or more (2, 3, or 4 more) pyrrole, pyrrole-like, and/orpyrroline subunits. The highly conjugated heterocyclic ring complexed toan iron ion is referred to herein as an iron complex. In someembodiments, the heme moiety can be a heme cofactor such as a hememoiety bound to a protein; a heme moiety bound to a non-proteinaceouspolymer; a heme moiety bound to a solid support; or a heme moietyencapsulated in a liposome. In some embodiments, the flavors and/oraromas are not generated in the absence of the iron complex (e.g., inthe absence of a ferrous chlorin) or are not generated in the absence ofa heme-cofactor (e.g., in the absence of a heme-containing protein).Accordingly, as described herein, the iron complexes such as isolatedchlorin-iron complexes or heme-cofactors (e.g., heme-containingproteins) can be used to generate meaty flavors and/or aromas in avariety of food products, such as during the cooking process.

Combining one or more iron complexes such as a heme-cofactor (e.g., aheme-containing protein, including, for example a plant-derived hemeprotein such as a plant leghemoglobin (legH)), with one or more flavorprecursor molecules or compositions (see, e.g., Table 1 or Table 13) cangenerate or provide a range of savory and meaty aromas and tastes (see,e.g., Tables 2, 7, and/or 11) in a cooked consumable food product.Flavor precursor molecules or compositions can be added to the uncookedfood product in purified form and/or can be derived from ingredients inthe uncooked consumable food product that contain and/or are enrichedwith one or more of the particular flavor precursors or compositions,including, for example, yeast extract, vegetable oil, corn oil, soybeanoil, palm fruit oil, palm kernel oil, safflower oil, flaxseed oil, ricebran oil, cottonseed oil, olive oil, canola oil, sunflower oil, coconutoil, mango oil, or an algal oil. The resultant flavor and/or aromaprofile can be modulated by the type and concentration of the flavorprecursors, the pH of the reaction, the length of cooking, the type andamount of iron complex (e.g., a heme cofactor such as a heme-containingprotein), the temperature of the reaction, and the amount of wateractivity in the product, among other factors.

One or more flavor precursor molecules or compositions can be addedalong with a iron complex (e.g., ferrous chlorophyllin or a hemecofactor such as a heme-containing protein), to an uncooked foodproduct, before and/or during the cooking process, to give the cookedconsumable food product a particular meaty taste and smell, for example,the taste and smell of beef, bacon, pork, lamb, or chicken. Consumablefood products can be animal or non-animal based (e.g., plant) foodproducts, or combinations of an animal and non-animal based foodproduct. For example, a plant based veggie burger or an animal-basedburger, such as a chicken burger, can be modified with the compositionsand methods of the present disclosure to result in a burger having acooked flavor and/or aroma profile that is more meat like, e.g.,beef-like, lamb-like, pork-like, turkey-like, duck-like, deer-like,yak-like, bison-like or other desirable meat flavor.

Food products for use in the present disclosure include those that havean iron-complex (e.g., a heme cofactor such as a heme-containingprotein), and one or more flavor precursor molecules included therein.The iron-complex such as a heme cofactor (e.g., a heme-containingprotein) and the one or more flavor precursor molecules can behomogenously or heterogeneously included in the food products. A hemeprotein can be isolated and purified prior to inclusion in the foodproduct. Non-limiting examples of consumable food products which caninclude an iron complex such as a heme-cofactor (e.g., a heme-containingprotein) and one or more flavor precursor molecules include animal-basedor non-animal (e.g., plant-based), or combinations of animal-based andnon-animal-based, food products in the form of hot dogs, burgers, groundmeat, sausages, steaks, filets, roasts, breasts, thighs, wings,meatballs, meatloaf, bacon, strips, fingers, nuggets, cutlets, or cubes.

Consumable food products for use in the present disclosure can be flavoradditive compositions, e.g., for addition to another consumable foodproduct before, during, or after its cooking process. A flavor additivecomposition can include an iron complex such as a heme-cofactor (e.g., aheme-containing protein), and one or more flavor precursors.

A flavor additive composition can include a heme protein, e.g., anisolated and purified heme protein; such a flavor additive compositioncan be used to modulate the flavor and/or aroma profile of a consumablefood product that comprises one or more flavor precursor molecules orcompositions. A flavor additive composition can include one or moreflavor precursor molecules or compositions; such a flavor additivecomposition can be used to modulate the flavor and/or aroma profile of aconsumable food product that comprises the heme protein, e.g., anisolated and purified heme protein.

A flavor additive composition can be in the form, of but not limited to,soup or stew bases, bouillon, e.g., powder or cubes, flavor packets, orseasoning packets or shakers. Such flavor additive compositions can beused to modulate the flavor and/or aroma profile for a variety ofconsumable food products, and can be added to a consumable food productbefore, during, or after cooking of the consumable food product.

In some embodiments, a flavor additive composition such as one includingan iron complex (e.g., ferrous chlorin or a heme protein) and one ormore flavor precursors can be reacted (e.g., in vitro) with heating togenerate a particular flavor and/or aroma profile of interest and theresultant product mixture can be added to the consumable food product ofinterest, which can then be eaten as-is or can be additionally modified,e.g., by additional cooking. In some embodiments, the iron complex canbe removed from the resultant product mixture before adding the productmixture to the consumable food product of interest. For example, theiron complex can be removed from the product mixture usingchromatographic techniques such as column chromatography, e.g., a columncontaining heme or iron-chlorin.

In some embodiments, the iron complex such as a heme-cofactor, e.g., aheme-protein, and the one or more flavor precursor flavor additivecompositions can be soy-free, wheat-free, yeast-free, MSG-free, and freeof protein hydrolysis products, and can taste meaty, highly savory, andwithout off odors or flavors.

In one aspect, this document features a food product that includes aniron complex such as a heme moiety, or a porphyrin, a porphyrinogen, acorrin, a corrinoid, a chlorin, a bacteriochorophyll, a corphin, achlorophyllin, a bacteriochlorin, or an isobacteriochlorin moietycomplexed to an iron ion and one or more flavor precursor moleculesselected from the group consisting of glucose, fructose, ribose,arabinose, glucose-6-phosphate, fructose 6-phosphate, fructose1,6-diphosphate, inositol, maltose, sucrose, maltodextrin, glycogen,nucleotide-bound sugars, molasses, a phospholipid, a lecithin, inosine,inosine monophosphate (IMP), guanosine monophosphate (GMP), pyrazine,adenosine monophosphate (AMP), lactic acid, succinic acid, glycolicacid, thiamine, creatine, pyrophosphate, vegetable oil, algal oil, cornoil, soybean oil, palm fruit oil, palm kernel oil, safflower oil,flaxseed oil, rice bran oil, cottonseed oil, sunflower oil, canola oil,olive oil, a free fatty acid, cysteine, methionine, isoleucine, leucine,lysine, phenylalanine, threonine, tryptophan, valine, arginine,histidine, alanine, asparagine, aspartate, glutamate, glutamine,glycine, proline, serine, tyrosine, glutathione, an amino acidderivative, a protein hydrolysate, a malt extract, a yeast extract, anda peptone. The heme moiety can be a heme-containing protein, a hememoiety bound to a non-peptidic polymer; or a heme moiety bound to asolid support. The heme-containing protein can be a plant, mammalian, ayeast or filamentous fungi, or bacterial heme-containing protein. Thefood product can include two to one hundred, two to fifty flavorprecursors, two to forty flavor precursors, two to thirty-five flavorprecursors, two to ten flavor precursors, or two to six flavorprecursors. In some embodiments, the one or more flavor precursormolecules are selected from the group consisting of glucose, ribose,cysteine, a cysteine derivative, thiamine, alanine, methionine, lysine,a lysine derivative, glutamic acid, a glutamic acid derivative, IMP,GMP, lactic acid, maltodextrin, creatine, alanine, arginine, asparagine,aspartate, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, methionine, phenylalanine, proline, threonine, tryptophan,tyrosine, valine, linoleic acid, and mixtures thereof. Theheme-containing protein can be a non-symbiotic hemoglobin or aleghemoglobin (e.g., a plant leghemoglobin such as one from soybean,alfalfa, lupin, pea, cow pea, or lupin). The heme-containing protein caninclude an amino acid sequence having at least 80% sequence identity toa polypeptide set forth in SEQ ID NOs:1-26. The heme-containing proteincan be isolated and purified. The food product further can include afood-grade oil, a seasoning agent, a flavoring agent, a protein, aprotein concentrate, an emulsifier, a gelling agent, or a fiber. Thefood product can be a meat substitute, a soup base, stew base, snackfood, bouillon powder, bouillon cube, a flavor packet, or a frozen foodproduct. Any of the food products can be free of animal products. Thefood product can be sealed within a packet or shaker.

This document also features a method for producing a flavor compound.The method can include combining an iron complex (e.g., a heme moiety, aporphyrin, a porphyrinogen, a corrin, a corrinoid, a chlorin, abacteriochorophyll, a corphin, a chlorophyllin, a bacteriochlorin, or anisobacteriochlorin complexed to an iron) and one or more flavorprecursor molecules to form a mixture, the one or more flavor precursormolecules selected from the group consisting of glucose, fructose,arabinose, ribose glucose-6-phosphate, fructose 6-phosphate, fructose1,6-diphosphate, inositol, maltose, sucrose, maltodextrin, glycogen,nucleotide-bound sugars, molasses, a phospholipid, a lecithin, inosine,inosine monophosphate (IMP), guanosine monophosphate (GMP), pyrazine,adenosine monophosphate (AMP), lactic acid, succinic acid, glycolicacid, thiamine, creatine, pyrophosphate, vegetable oil, algal oil, cornoil, soybean oil, palm fruit oil, palm kernel oil, safflower oil,flaxseed oil, rice bran oil, cottonseed oil, canola oil, olive oil,sunflower oil, flaxseed oil, coconut oil, mango oil, a free fatty acid,cysteine, methionine, isoleucine, leucine, lysine, phenylalanine,threonine, tryptophan, valine, arginine, histidine, alanine, asparagine,aspartate, glutamate, glutamine, glycine, proline, serine, tyrosine,glutathione, an amino acid derivative, a protein hydrolysate, a maltextract, a yeast extract, and a peptone; and heating the mixture to formone or more flavor compounds selected from the group consisting ofphenylacetaldehyde, 1-octen-3-one, 2-n-heptylfuran,2-thiophenecarboxaldehyde, 3-thiophenecarboxaldehyde, butyrolactone,2-undecenal, pyrazine, methyl-, furfural, 2-decanone, pyrrole,1-octen-3-ol, 2-acetylthiazole, (E)-2-octenal, decanal, benzaldehyde,(E)-2-nonenal, pyrazine, 1-hexanol, 1-heptanol, dimethyl trisulfide,2-nonanone, 2-pentanone, 2-heptanone, 2,3-butanedione, heptanal,nonanal, 2-octanone, 1-octanol, 3-ethylcyclopentanone, 3-octen-2-one,(E,E)-2,4-heptadienal, (Z)-2-heptenal, 2-heptanone, 6-methyl-,(Z)-4-heptenal, (E,Z)-2,6-nonadienal, 3-methyl-2-butenal,2-pentyl-furan, thiazole, (E, E)-2,4-decadienal, hexanoic acid,1-ethyl-5-methylcyclopentene, (E,E)-2,4-nonadienal, (Z)-2-decenal,dihydro-5-pentyl-2(3H)-furanone, trans-3-nonen-2-one,(E,E)-3,5-octadien-2-one, (Z)-2-octen-1-ol,5-ethyldihydro-2(3H)-furanone, 2-butenal, 1-penten-3-ol, (E)-2-hexenal,formic acid, heptyl ester, 2-pentyl-thiophene, (Z)-2-nonenal,2-hexyl-thiophene, (E)-2-decenal, 2-ethyl-5-methyl-pyrazine,3-ethyl-2,5-dimethyl-pyrazine, 2-ethyl-1-hexanol, thiophene,2-methyl-furan, pyridine, butanal, 2-ethyl-furan, 3-methyl-butanal,trichloromethane, 2-methyl-butanal, methacrolein, 2-methyl-propanal,propanal, acetaldehyde, 2-propyl-furan, dihydro-5-propyl-2(3H)-furanone,1,3-hexadiene, 4-decyne, pentanal, 1-propanol, heptanoic acid,trimethyl-ethanethiol, 1-butanol, 1-penten-3-one, dimethyl sulfide,2-ethyl furan, 2-pentyl-thiophene, 2-propenal, 2-tridecen-1-ol,4-octene, 2-methyl thiazole, methyl-pyrazine, 2-butanone,2-pentyl-furan, 2-methyl-propanal, butyrolactone, 3-methyl-butanal,methyl-thiirane, 2-hexyl-furan, butanal, 2-methyl-butanal,2-methyl-furan, furan, octanal, 2-heptenal, 1-octene, formic acid heptylester, 3-pentyl-furan, and 4-penten-2-one. The heme moiety can be aheme-containing protein, a heme moiety bound to a non-peptidic polymer;or a heme moiety bound to a solid support. The method can includecombining cysteine, ribose, lactic acid, lysine, and/or thiamine withthe heme-containing protein.

In another aspect, this document features a method for producing aflavor compound. The method includes combining an iron complex, such asa heme-containing protein, and one or more flavor precursor molecules toform a mixture, the one or more flavor precursor molecules selected fromthe group consisting of glucose, fructose, ribose, arabinose,glucose-6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate,inositol, maltose, sucrose, maltodextrin, glycogen, nucleotide-boundsugars, molasses, a phospholipid, a lecithin, inosine, IMP, GMP,pyrazine, AMP, lactic acid, succinic acid, glycolic acid, thiamine,creatine, pyrophosphate, vegetable oil, algal oil, corn oil, soybeanoil, palm fruit oil, palm kernel oil, safflower oil, flaxseed oil, ricebran oil, cottonseed oil, olive oil, sunflower oil, canola oil, flaxseedoil, coconut oil, mango oil, a free fatty acid, methionine, cysteine,isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan,valine, arginine, histidine, alanine, asparagine, aspartate, glutamate,glutamine, glycine, proline, serine, tyrosine, glutathione, an aminoacid derivative, a protein hydrolysate, a malt extract, a yeast extract,and a peptone; and heating the mixture to form one or more flavorcompounds set forth in Tables 3, 8, or 9. For example, the flavorprecursors can include cysteine, a sugar, and one or more otherprecursors.

This document also features a method for imparting a meat like flavor(e.g., beef-like, chicken like, pork-like, lamb-like, turkey-like,duck-like, deer-like, or bison-like) to a food product. The methodincludes contacting the food product with a flavoring composition, theflavoring composition comprising i) an iron complex, such as a hememoiety (e.g., a heme-containing protein); and ii) one or more flavorprecursor molecules selected from the group consisting of glucose,fructose, ribose, arabinose, glucose-6-phosphate, fructose 6-phosphate,fructose 1,6-diphosphate, inositol, maltose, sucrose, maltodextrin,glycogen, nucleotide-bound sugars, molasses, a phospholipid, a lecithin,inosine, IMP, GMP, pyrazine, AMP, lactic acid, succinic acid, glycolicacid, thiamine, creatine, pyrophosphate, vegetable oil, algal oil, cornoil, soybean oil, palm fruit oil, palm kernel oil, safflower oil,flaxseed oil, rice bran oil, cottonseed oil, olive oil, sunflower oil,canola oil, flaxseed oil, coconut oil, mango oil, a free fatty acid,cysteine, methionine, isoleucine, leucine, lysine, phenylalanine,threonine, tryptophan, valine, arginine, histidine, alanine, asparagine,aspartate, glutamate, glutamine, glycine, proline, serine, tyrosine,glutathione, an amino acid derivative, a protein hydrolysate, a maltextract, a yeast extract, and a peptone; wherein after heating the foodproduct and the flavoring composition together, a meat like flavor(e.g., beef-like, chicken like, pork-like, lamb-like, turkey-like,duck-like, deer-like, or bison-like) is imparted to the food product. Insome embodiments, the iron complex is removed from the food product. Theflavoring composition further can include a seasoning agent, a flavoringagent, a protein, a protein concentrate, or an emulsifier. The flavoringcomposition can be sealed within a packet or shaker.

In another aspect, this document features a method of making a foodproduct. The method includes combining an isolated heme-containingprotein and one or more flavor precursor molecules to form a mixture,the one or more flavor precursor molecules selected from the groupconsisting of glucose, fructose, ribose, arabinose, glucose-6-phosphate,fructose 6-phosphate, fructose 1,6-diphosphate, inositol, maltose,sucrose, maltodextrin, glycogen, nucleotide-bound sugars, molasses, aphospholipid, a lecithin, inosine, IMP, GMP, pyrazine, AMP, lactic acid,succinic acid, glycolic acid, thiamine, creatine, pyrophosphate,sunflower oil, coconut oil, canola oil, flaxseed oil, mango oil, a freefatty acid, cysteine, methionine, isoleucine, leucine, lysine,phenylalanine, threonine, tryptophan, valine, arginine, histidine,alanine, asparagine, aspartate, glutamate, glutamine, glycine, proline,serine, tyrosine, glutathione, an amino acid derivative, a proteinhydrolysate, a malt extract, a yeast extract, and a peptone; and heatingthe mixture.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims. The word “comprising” inthe claims may be replaced by “consisting essentially of” or with“consisting of,” according to standard practice in patent law.

DESCRIPTION OF THE DRAWINGS

FIG. 1 contains amino acid sequences of exemplary heme-containingproteins.

FIG. 2 is a bar graph of the beefiness rating of the meat replica withor without the Magic Mix, both samples in triplicate with 1% w/v LegHprotein. Tasters rated beefiness on a scale from 1-7, with 1 being notbeefy at all and 7 being exactly like ground beef.

DETAILED DESCRIPTION

This document is based on methods and materials for modulating the tasteand/or aroma profile of food products. As described herein, compositionscontaining one or more flavor precursors and one or more highlyconjugated heterocyclic rings complexed to an iron (referred to hereinas an iron complex) can be used to modulate the taste and/or aromaprofile of food products. Such iron complexes include heme moieties orother highly conjugated heterocylic rings complexed to an iron ion(referred to as an iron complex). “Heme” refers to a prosthetic groupbound to iron (Fe′ or Fe′) in the center of a porphyrin ring. Thus, aniron complex can be a heme moiety, or a porphyrin, porphyrinogen,corrin, corrinoid, chlorin, bacteriochorophyll, corphin, chlorophyllin,bacteriochlorin, or isobacteriochlorin moiety complexed to iron ion. Theheme moiety that can be used to modulate the taste and/or aroma profileof food products can be a heme cofactor such as a heme-containingprotein; a heme moiety bound to a non-peptidic polymer or othermacromolecule such as a liposome, a polyethylene glycol, a carbohydrate,a polysaccharide, a cyclodextrin, a polyethylenimine, a polyacrylate, orderivatives thereof; a siderophore (i.e., an iron chelating compound);or a heme moiety bound to a solid support (e.g., beads) composed of achromatography resin, cellulose, graphite, charcoal, or diatomaceousearth.

In some embodiments, the iron complexes catalyze some reactions andproduce flavor precursors without heating or cooking. In someembodiments, the iron complex destabilizes upon heating or cooking andreleases the iron, e.g., the protein is denatured, so flavor precursorscan be generated.

Suitable flavor precursors include sugars, sugar alcohols, sugarderivatives, oils (e.g., vegetable oils), free fatty acids,alpha-hydroxy acids, dicarboxylic acids, amino acids and derivativesthereof, nucleosides, nucleotides, vitamins, peptides, proteinhydrolysates, extracts, phospholipids, lecithin, and organic molecules.Non-limiting examples of such flavor precursors are provided in Table 1.

TABLE 1 Flavor Precursor Molecules   Sugars, sugar alcohols, sugaracids, and sugar derivatives: glucose, fructose, ribose, sucrose,arabinose, glucose-6-phosphate,fructose-6-phosphate, fructose1,6-diphosphate, inositol, maltose, molasses, maltodextrin, glycogen,galactose, lactose, ribitol, gluconic acid and glucuronic acid, amylose,amylopectin, or xylose Oils: coconut oil, mango oil, sunflower oil,cottonseed oil, safflower oil, rice bran oil, cocoa butter, palm fruitoil, palm oil, soybean oil, canola oil, corn oil, sesame oil, walnutoil, flaxseed, jojoba oil, castor, grapeseed oil, peanut oil, olive oil,algal oil, oil from bacteria or fungi Free fatty acids: caprylic acid,capric acid, lauric acid, myristic acid, palmititic acid, palmitoleicacid, stearic, oleic acid, linoleic acid, alpha linolenic acid, gammalinolenic acid, arachidic acid, arachidonic acid, behenic acid, orerucic acid Amino acids and derivatives thereof: cysteine, cystine, acysteine sulfoxide, allicin, selenocysteine, methionine, isoleucine,leucine, lysine, phenylalanine, threonine, tryptophan,5-hydroxytryptophan, valine, arginine, histidine, alanine, asparagine,aspartate, glutamate, glutamine, glycine, proline, serine, or tyrosineNucleosides and Nucleotides: inosine, inosine monophosphate (IMP),guanosine, guanoside monophosphate (GMP), adenosine, adenosinemonophosophate (AMP) Vitamins: thiamine, vitamin C, Vitamin D, VitaminB6, or Vitamin E Misc: phospholipid, lecithin, pyrazine, creatine,pyrophosphate Acids: acetic acid, alpha hydroxy acids such as lacticacid or glycolic acid, tricarboxylic acids such as citric acid,dicarboxylic acids such as succinic acid or tartaric acid Peptides andprotein hydrolysates: glutathione, vegetable protein hydrolysates, soyprotein hydrolysates, yeast protein hydrolysates, algal proteinhydrolysatess, meat protein hydrolysates Extracts: a malt extract, ayeast extract, and a peptone

In some embodiments, one flavor precursor or combinations of two to onehundred flavor precursors, two to ninety, two to eighty, two to seventy,two to sixty, or two to fifty flavor precursors are used. For example,combinations of two to forty flavor precursors, two to thirty-fiveflavor precursors, two to ten flavor precursors, or two to six flavorprecursors can be used with the one or more iron complexes (e.g., hemeco-factors such as a heme-containing proteins). For example, the one ormore flavor precursors can be glucose, ribose, cysteine, a cysteinederivative, thiamine, lysine, a lysine derivative, glutamic acid, aglutamic acid derivative, alanine, methionine, IMP, GMP, lactic acid,and mixtures thereof (e.g., glucose and cysteine; cysteine and ribose;cysteine, glucose or ribose, and thiamine; cysteine, glucose or ribose,IMP, and GMP; cysteine, glucose or ribose, and lactic acid). Forexample, the one or more flavor precursors can be alanine, arginine,asparagine, aspartate, cysteine, glutamic acid, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, threonine, tryptophan, tyrosine, valine, glucose, ribose,maltodextrin, thiamine, IMP, GMP, lactic acid, and creatine.

As used herein, the term “heme containing protein” can be usedinterchangeably with “heme containing polypeptide” or “heme protein” or“heme polypeptide” and includes any polypeptide that can covalently ornoncovalently bind a heme moiety. In some embodiments, theheme-containing polypeptide is a globin and can include a globin fold,which comprises a series of seven to nine alpha helices. Globin typeproteins can be of any class (e.g., class I, class II, or class III),and in some embodiments, can transport or store oxygen. For example, aheme-containing protein can be a non-symbiotic type of hemoglobin or aleghemoglobin. A heme-containing polypeptide can be a monomer, i.e., asingle polypeptide chain, or can be a dimer, a trimer, tetramer, and/orhigher order oligomers. The life-time of the oxygenated Fe2⁺ state of aheme-containing protein can be similar to that of myoglobin or canexceed it by 10%, 20%, 30% 50%, 100% or more under conditions in whichthe heme-protein-containing consumable is manufactured, stored, handledor prepared for consumption. The life-time of the unoxygenated Fe²⁺state of a heme-containing protein can be similar to that of myoglobinor can exceed it by 10%, 20%, 30% 50%, 100% or more under conditions inwhich the heme-protein-containing consumable is manufactured, stored,handled or prepared for consumption

Non-limiting examples of heme-containing polypeptides can include anandroglobin, a cytoglobin, a globin E, a globin X, a globin Y, ahemoglobin, a myoglobin, an erythrocruorin, a beta hemoglobin, an alphahemoglobin, a protoglobin, a cyanoglobin, a cytoglobin, a histoglobin, aneuroglobins, a chlorocruorin, a truncated hemoglobin (e.g., HbN orHbO), a truncated 2/2 globin, a hemoglobin 3 (e.g., Glb3), a cytochrome,or a peroxidase.

Heme-containing proteins that can be used in the compositions and foodproducts described herein can be from mammals (e.g., farms animals suchas cows, goats, sheep, pigs, ox, or rabbits), birds, plants, algae,fungi (e.g., yeast or filamentous fungi), ciliates, or bacteria. Forexample, a heme-containing protein can be from a mammal such as a farmanimal (e.g., a cow, goat, sheep, pig, ox, or rabbit) or a bird such asa turkey or chicken. Heme-containing proteins can be from a plant suchas Nicotiana tabacum or Nicotiana sylvestris (tobacco); Zea mays (corn),Arabidopsis thaliana, a legume such as Glycine max (soybean), Cicerarietinum (garbanzo or chick pea), Pisum sativum (pea) varieties such asgarden peas or sugar snap peas, Phaseolus vulgaris varieties of commonbeans such as green beans, black beans, navy beans, northern beans, orpinto beans, Vigna unguiculata varieties (cow peas), Vigna radiata (Mungbeans), Lupinus albus (lupin), or Medicago sativa (alfalfa); Brassicanapus (canola); Triticum sps. (wheat, including wheat berries, andspelt); Gossypium hirsutum (cotton); Oryza sativa (rice); Zizania sps.(wild rice); Helianthus annuus (sunflower); Beta vulgaris (sugarbeet);Pennisetum glaucum (pearl millet); Chenopodium sp. (quinoa); Sesamum sp.(sesame); Linum usitatissimum (flax); or Hordeum vulgare (barley).Heme-containing proteins can be isolated from fungi such asSaccharomyces cerevisiae, Pichia pastoris, Magnaporthe oryzae, Fusariumgraminearum, Aspergillus oryzae, Trichoderma reesei, Mycelioptherathermophile, Kluyvera lactis, or Fusarium oxysporum. Heme-containingproteins can be isolated from bacteria such as Escherichia coli,Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium,Synechocistis sp., Aquifex aeolicus, Methylacidiphilum infernorum, orthermophilic bacteria such as Thermophilus. The sequences and structureof numerous heme-containing proteins are known. See for example, Reedy,et al., Nucleic Acids Research, 2008, Vol. 36, Database issue D307-D313and the Heme Protein Database available on the world wide web athttp://hemeprotein.info/heme.php.

For example, a non-symbiotic hemoglobin can be from a plant selectedfrom the group consisting of soybean, sprouted soybean, alfalfa, goldenflax, black bean, black eyed pea, northern, garbanzo, moong bean,cowpeas, pinto beans, pod peas, quinoa, sesame, sunflower, wheatberries, spelt, barley, wild rice, or rice.

Any of the heme-containing proteins described herein that can be usedfor producing food products can have at least 70% (e.g., at least 75%,80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) sequence identity to theamino acid sequence of the corresponding wild-type heme-containingprotein or fragments thereof that contain a heme-binding motif. Forexample, a heme-containing protein can have at least 70% sequenceidentity to an amino acid sequence set forth in FIG. 1, including anon-symbiotic hemoglobin such as that from Vigna radiata (SEQ ID NO:1),Hordeum vulgare (SEQ ID NO:5), Zea mays (SEQ ID NO:13), Oryza sativasubsp. japonica (rice) (SEQ ID NO:14), or Arabidopsis thaliana (SEQ IDNO:15), a Hell's gate globin I such as that from Methylacidiphiluminfernorum (SEQ ID NO:2), a flavohemoprotein such as that from Aquifexaeolicus (SEQ ID NO:3), a leghemoglobin such as that from Glycine max(SEQ ID NO:4), Pisum sativum (SEQ ID NO:16), or Vigna unguiculata (SEQID NO:17), a heme-dependent peroxidase such as from Magnaporthe oryzae,(SEQ ID NO:6) or Fusarium oxysporum (SEQ ID NO:7), a cytochrome cperoxidase from Fusarium graminearum (SEQ ID NO:8), a truncatedhemoglobin from Chlamydomonas moewusii (SEQ ID NO:9), Tetrahymenapyriformis (SEQ ID NO:10, group I truncated), Paramecium caudatum (SEQID NO:11, group I truncated), a hemoglobin from Aspergillus niger (SEQID NO:12), or a mammalian myoglobin protein such as the Bos taurus (SEQID NO:18) myoglobin, Sus scrofa (SEQ ID NO:19) myoglobin, Equus caballus(SEQ ID NO:20) myoglobin, a heme-protein from Nicotiana benthamiana (SEQID NO:21), Bacillus subtilis (SEQ ID NO:22), Corynebacterium glutamicum(SEQ ID NO:23), Synechocystis PCC6803 (SEQ ID NO:24), Synechococcus sp.PCC 7335 (SEQ ID NO:25), or Nostoc commune (SEQ ID NO:26).

The percent identity between two amino acid sequences can be determinedas follows. First, the amino acid sequences are aligned using the BLAST2 Sequences (Bl2seq) program from the stand-alone version of BLASTZcontaining BLASTP version 2.0.14. This stand-alone version of BLASTZ canbe obtained from Fish & Richardson's web site (e.g., www.fr.com/blast/)or the U.S. government's National Center for Biotechnology Informationweb site (www.ncbi.nlm.nih.gov). Instructions explaining how to use theBl2seq program can be found in the readme file accompanying BLASTZ.Bl2seq performs a comparison between two amino acid sequences using theBLASTP algorithm. To compare two amino acid sequences, the options ofBl2seq are set as follows: -i is set to a file containing the firstamino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to afile containing the second amino acid sequence to be compared (e.g.,C:\seq2.txt); -p is set to blastp; -o is set to any desired file name(e.g., C:\output.txt); and all other options are left at their defaultsetting. For example, the following command can be used to generate anoutput file containing a comparison between two amino acid sequences:C:\Bl2seq c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If thetwo compared sequences share homology, then the designated output filewill present those regions of homology as aligned sequences. If the twocompared sequences do not share homology, then the designated outputfile will not present aligned sequences. Similar procedures can befollowing for nucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity is determined by dividing the number ofmatches by the length of the full-length polypeptide amino acid sequencefollowed by multiplying the resulting value by 100. It is noted that thepercent identity value is rounded to the nearest tenth. For example,78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15,78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It also is notedthat the length value will always be an integer.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid. For example, codons in the coding sequence for a givenenzyme can be modified such that optimal expression in a particularspecies (e.g., bacteria or fungus) is obtained, using appropriate codonbias tables for that species.

Heme-containing proteins can be extracted from the source material(e.g., extracted from animal tissue, or plant, fungal, algal, orbacterial biomass, or from the culture supernatant for secretedproteins) or from a combination of source materials (e.g., multipleplant species). Leghemoglobin is readily available as an unusedby-product of commodity legume crops (e.g., soybean, alfalfa, or pea).The amount of leghemoglobin in the roots of these crops in the UnitedStates exceeds the myoglobin content of all the red meat consumed in theUnited States.

In some embodiments, extracts of heme-containing proteins include one ormore non-heme-containing proteins from the source material (e.g., otheranimal, plant, fungal, algal, or bacterial proteins) or from acombination of source materials (e.g., different animal, plant, fungi,algae, or bacteria).

In some embodiments, heme-containing proteins are isolated and purifiedfrom other components of the source material (e.g., other animal, plant,fungal, algal, or bacterial proteins). As used herein, the term“isolated and purified” indicates that the preparation ofheme-containing protein is at least 60% pure, e.g., greater than 65%,70%, 75%, 80%, 85%, 90%, 95%, or 99% pure. Without being bound bytheory, isolating and purifying proteins can allow the food products tobe made with greater consistency and greater control over the propertiesof the food product as unwanted material is eliminated. Proteins can beseparated on the basis of their molecular weight, for example, by sizeexclusion chromatography, ultrafiltration through membranes, or densitycentrifugation. In some embodiments, the proteins can be separated basedon their surface charge, for example, by isoelectric precipitation,anion exchange chromatography, or cation exchange chromatography.Proteins also can be separated on the basis of their solubility, forexample, by ammonium sulfate precipitation, isoelectric precipitation,surfactants, detergents or solvent extraction. Proteins also can beseparated by their affinity to another molecule, using, for example,hydrophobic interaction chromatography, reactive dyes, orhydroxyapatite. Affinity chromatography also can include usingantibodies having specific binding affinity for the heme-containingprotein, nickel NTA for His-tagged recombinant proteins, lectins to bindto sugar moieties on a glycoprotein, or other molecules whichspecifically binds the protein.

Heme-containing proteins also can be recombinantly produced usingpolypeptide expression techniques (e.g., heterologous expressiontechniques using bacterial cells, insect cells, fungal cells such asyeast, plant cells such as tobacco, soybean, or Arabidopsis, ormammalian cells). In some cases, standard polypeptide synthesistechniques (e.g., liquid-phase polypeptide synthesis techniques orsolid-phase polypeptide synthesis techniques) can be used to produceheme-containing proteins synthetically. In some cases, in vitrotranscription-translation techniques can be used to produceheme-containing proteins.

The protein used in the consumable may be soluble in a solution. In someembodiments, the isolated and purified proteins are soluble in solutionat greater than 5, 10, 15, 20, 25, 50, 100, 150, 200, or 250 g/L.

In some embodiments, the isolated and purified protein is substantiallyin its native fold and water soluble. In some embodiments, the isolatedand purified protein is more than 50, 60, 70, 80, or 90% in its nativefold. In some embodiments, the isolated and purified protein is morethan 50, 60, 70, 80, or 90% water soluble.

Modulating Flavor and/or Aroma Profiles

As described herein, different combinations of flavor precursors can beused with one or more iron complexes (e.g., a ferrous chlorin, achlorin-iron complex, or a heme-cofactor such as a heme-containingprotein or heme bound to a non-peptidic polymer such as polyethyleneglycol or to a solid support) to produce different flavor and aromaprofiles when the flavor precursors and iron complexes are heatedtogether (e.g., during cooking). The resultant flavor and/or aromaprofile can be modulated by the type and concentration of the flavorprecursors, the pH of the reaction, the length of cooking, the type andamount of iron complex (e.g., a heme-cofactor such as heme-containingprotein, heme bound to non-peptidic polymer or macromolecule, or hemebound to a solid support), the temperature of the reaction, and theamount of water activity in the product, among other factors. Inembodiments in which a heme moiety is bound to a solid support such ascellulose or a chromatography resin, graphite, charcoal, or diatomaceousearth, the solid support (e.g., beads) can be incubated with sugarsand/or one or more other flavor precursors to generate flavors, and thenthe solid support with attached heme moiety can be re-used, i.e.,incubated again with sugars and/or one or more other flavor precursorsto generate flavors.

Table 2 provides non-limiting examples of flavor types that can begenerated by combining one or more flavor precursors and one or moreheme co-factors (e.g., heme-containing proteins). See also Tables 7and/or 11.

TABLE 2 Flavor Types beef beef broth beef dripping cheesy cold-cut delimeat squash bacon sharp meaty fruity brothy floral ramen musty egg friedfood malty caramel bready barbeque sulfur chocolate fried chicken sweetbrowned potato pretzel french toast grassy breadcrust bloody mushroombroccoli chicken brothy cumin buttery umami metallic raisin yeasty goatyvegetable broth

Flavor and aroma profiles are created by different chemical compoundsformed by chemical reactions between the heme co-factor (e.g.,heme-containing protein) and flavor precursors. Gas chromatography-massspectrometry (GCMS) can be used to separate and identify the differentchemical compounds within a test sample. For example, volatile chemicalscan be isolated from the head space after heating a heme-containingprotein and one or more flavor precursors.

Table 3 provides non-limiting examples of compounds that can beproduced. See also Tables 8, 9, 12, and/or 14.

TABLE 3 Compounds Produced phenylacetaldehyde 2-butenal, 2-ethyl-1,3-hexadiene 1-octen-3-one acetonitrile 4-decyne 2-n-heptylfuranpentanal 2-thiophenecarboxaldehyde (E)-2-Hexenal 1-propanol3-thiophenecarboxaldehyde 4-ethyl-phenol, heptanoic acid 1-octene3-octanone ethanethiol butyrolactone styrene 2-methyl-1-heptene2-undecenal furan, 3-pentyl- (E)-4-octene propyl-cyclopropane formicacid, heptyl ester 2-methyl-2-heptene methyl-pyrazine (E)-2-Heptenalpentanoic acid 1-hydroxy-propanone 6-methyl-5-hepten-2-one nonanoic acidacetic acid n-caproic acid vinyl ester 1,3-dimethyl-benzene furfural2-ethyl-2-hexenal 2-decanone 1-hepten-3-ol toluene pyrrole1-ethyl-1-methyl- 1-butanol cyclopentane 1-octen-3-ol3-ethy1-2-methy1-1,3- 2,3,3-trimethyl-pentane hexadiene 2-acetylthiazole2-pentyl-thiophene isopropyl alcohol (E)-2-octenal (Z)-2-nonenal2,2,4,6,6-pentamethyl- heptane decanal 2-n-octylfuran phenolbenzaldehyde 2-hexyl-thiophene 1-penten-3-one (E)-2-Nonenal4-cyclopentene-1,3-dione dimethyl sulfide pyrazine 1-nonanol thiirane1-pentanol (E)-2-decenal (E)-2-octen-1-ol trans-2-(2-pentenyl)furan4-ethyl-benzaldehyde 2,4-dimethyl-1-heptene 1-hexanol 1,7-octadien-3-ol1,3-bis(1,1- dimethylethyl)-benzene 1-heptanol octanoic acid heptanedimethyl trisulfide 2-ethyl-5-methyl-pyrazine 4,7-dimethyl-undecane2-nonanone 3-ethyl-2,5-dimethyl- acetophenone pyrazine 2-pentanone1,3,5-cycloheptatriene tridecane 2-heptanone 2-ethyl-1-hexanolthiophosphoramide, s- methyl ester 2,3-butanedione 4-methyl-octanoicacid 2-methyl-thiazole heptanal m-aminophenylacetylene3-(1-methylethoxy)- propanenitrile, nonanal benzene 2,4-bis(1,1-dimethylethyl)-phenol 2-octanone thiophene 3-ethy1-2,2-dimethy1- pentane2-butanone 2-methyl-furan 3-ethyl-pentane octanal pyridine2,3,4-trimethyl-pentane 1-octanol furan 2,4,6-trimethyl-octane3-ethylcyclopentanone butanal 2,6-dimethyl-nonane 8-methyl-1-undecene2-ethyl-furan 2-hexyl-furan 3-octen-2-one carbon disulfide 4-methyl-5-thiazoleethanol 2,4-Heptadienal, (E,E)- Furan, 2-hexyl-:2 4-penten-2-one(Z)-2-heptenal 3-methyl-butanal 4-methylthiazole 6-methyl-2-heptanone2-methyl-butanal 2-methyl-3-pentanone (Z)-4-heptenal methacrolein2,3-pentanedione (E,Z)-2,6-nonadienal octane (E)-2-tridecen-1-ol3-methyl-2-butenal ethanol 2- thiophenemethanamine 2-pentyl-furan2-methyl-propanal (Z)-2-nonenal, thiazole acetone methyl thiolacetate(E,E)-2,4-decadienal propanal methyl ethanoate hexanoic acidmethyl-thiirane isothiazole 1-ethyl-5-methylcyclopentene acetaldehyde3,3-dimethyl-hexane (E,E)-2,4-nonadienal 2-propenal 4-methyl-heptane(Z)-2-decenal 2-propyl-furan 2,4-dimethyl-heptanedihydro-5-pentyl-2(3h)-furanone dihydro-5-propyl-2(3H)-2,3,4-trimethyl-heptane furanone trans-3-nonen-2-onedihydro-3-(2H)-thiophenone 2-methyl-heptane (E,E)-3,5-octadien-2-one2,2,6-trimethyl-decane 2-methyl-3-furanthiol (Z)-2-octen-1-ol3,3′-dithiobis[2-methyl- 4-amino-1,2,5- furan oxadiazole-3- carbonitrile5-ethyldihydro-2(3h)-furanone 1-heptene 1,2-benzisothiazol- 3(2H)-one2-butenal 1,3-octadiene 2-acetyl-propen-2-ol, 1-penten-3-ol 1-nonene1-decen-3-one 1-(ethylthio)-2-(methylthio)-buta-1,3- diene

In some embodiments, an iron complex (e.g., a ferrous chlorin or aheme-cofactor such as a heme-containing protein) described herein isheated in the presence of ground chicken, to increase specific volatileflavor and odorant components typically elevated in beef. For example,propanal, butanal, 2-ethyl-furan, heptanal, octanal,trans-2-(2-pentenyl)furan, (Z)-2-heptenal, (E)-2-octenal, pyrrole,2,4-dodecadienal, 1-octanal, (Z)-2-decenal, or 2-undecenal can beincreased in the presence of the heme-containing protein, which canimpart a more beefy flavor to the chicken.

In some embodiments, an iron complex (e.g., a ferrous chlorin or aheme-cofactor such as a heme-containing protein) described herein isheated in the presence of cysteine and glucose or other combinations offlavor precursors to provide a different profile of volatile odorantsthan when any subset of the three components are used individually.Volatile flavor components that are increased under these conditionsinclude but are not limited to furan, acetone, thiazole, furfural,benzaldehyde, 2-pyridinecarboxaldehyde,5-methyl-2-thiophenecarboxaldehyde, 3-methyl-2-thiophenecarboxaldehyde,3-thiophenemethanol and decanol. See, e.g., Tables 8 and 9. Under theseconditions, cysteine and glucose alone or in the presence of iron saltssuch as ferrous glucanate produced a sulfurous, odor, but addition ofheme-containing proteins reduced the sulfurous odor and replaced it withflavors including but not limited to chicken broth, burnt mushroom,molasses, and bread.

In some embodiments, an iron complex (e.g., a ferrous chlorin or aheme-cofactor such as a heme-containing protein) described herein isheated in the presence of cysteine and ribose to provide a differentprofile of volatile odorants. Heating in the presence of ribose createdsome additional compounds as compared to when a heme-containing proteinand glucose were heated together. See Tables 8 and 9.

In some embodiments, an iron complex (e.g., a ferrous chlorophillin or aheme-cofactor such as a heme-containing protein) described herein can beheated in the presence of thiamine and a sugar to affect the formationof 5-Thiazoleethanol, 4-methyl-furan, 3,3′-dithiobis[2-methyl-furan,and/or 4-Methylthiazole. These compounds are known to be present in meatand have beefy, meaty taste notes.

In some embodiments, an iron complex (e.g., a ferrous chlorin or aheme-cofactor such as a heme-containing protein) described herein can beheated in the presence of a nucleotide such as inosine monophosphateand/or guanosine monophosphate to control the formation of flavorcompounds such as (E)-4-octene, 2-ethyl-furan, 2-pentanone,2,3-butanedione, 2-methyl-thiazole, methyl-pyrazine, tridecane,(E)-2-octenal, 2-thiopenecarboxaldehyde, and/or3-thiopenecarboxaldehyde. These compounds are known to be present inmeat and have a beefy, meaty, buttery, and or savory flavor notes.

In some embodiments, an iron complex (e.g., a ferrous chlorin or aheme-cofactor such as a heme-containing protein) described herein can beheated in the presence of lysine, a sugar such as ribose, and cysteineto control the formation of flavor compounds such as dimethyltrisulfide, nonanal, 2-pentyl thiophene, 2-nonenal furfural, 1-octanol,2-nonenal, thiazole, 2-acetylthiazole, phenylacetaldehyde, and/or2-acetylthiazole. These compounds are known to be present in meat andsome have a beefy, meaty, and or savory flavor.

In some embodiments, an iron complex (e.g., a ferrous chlorin or aheme-cofactor such as a heme-containing protein) described herein can beheated in the presence of lactic acid, a sugar such as ribose, andcysteine to control the formation of the flavor compounds nonanal,thiazole, 2-acetylthiazole, and/or 8-methyl 1-undecene. These compoundsare known to be present in meat and have beefy, savory, browned, bready,and malty notes.

In some embodiments, an iron complex (e.g., a ferrous chlorin or aheme-cofactor such as a heme-containing protein) described herein can beheated in the presence of amino acids, sugars such as glucose, ribose,and maltodextrin, lactic acid, thiamine, IMP, GMP, creatine, and saltssuch as potassium chloride and sodium chloride, to control the formationof flavor compounds such as 1,3-bis(1,1-dimethylethyl)-benzene, 2-methyl3-furanthiol, and/or bis(2-methyl-4,5-dihydro-3-furyl) disulfide. Thesecompounds are known to be present in meat and have beefy notes. See alsoTable 14.

In some embodiments, a particular type of heme-containing protein ischosen to control the formation of flavor compounds. See, for example,the results of Table 9, which shows that the addition of different typesof heme-proteins (LegH, Barley, B. myoglobin, or A. aeolicus) in flavorreaction mixtures containing one or more flavor precursor compoundsresults in many of the same key meat flavors, including but not limitedto pentanone, 3-methyl butanal, 2-methyl butanal, 2-heptenal, 1-octene,nonanal, 2-propenal, 2-decenal, 2-nonanone, 2-octanone, 2-tridecen-1-ol,2-octanone, 2-octenal, 4-methyl-2-heptanone, octanal, 2-undecenal,butyrolactone, 1-octen-3-one, 3-methylheptyl acetate, and2-pentyl-thiophene. These differences in flavor compounds can change theoverall taste profile.

In some embodiments, an iron complex (e.g., a ferrous chlorin or aheme-cofactor such as a heme-containing protein) described herein andone or more flavor precursors can be reacted (e.g., in vitro) withheating to generate a particular flavor and/or aroma profile of interestand the resultant flavor additive composition can be added to theconsumable food product of interest, which can then be eaten as-is orcan be additionally modified, e.g., by additional cooking.

In some embodiments, any undesirable flavors can be minimized bydeodorizing with activated charcoal or by removing enzymes such aslipoxygenases (LOX), which can be present in trace amounts when usingpreparations of plant proteins, and which can convert unsaturatedtriacylglycerides (such as linoleic acid or linolenic acid) into smallerand more volatile molecules. LOX are naturally present in legumes suchas peas, soybeans, and peanuts, as well as rice, potatoes, and olives.When legume flours are fractionated into separate protein fractions, LOXcan act as undesirable “time-bombs” that can cause undesirable flavorson aging or storage. Compositions containing plant proteins (e.g., fromground plant seeds) can be subjected to purification to remove LOXusing, for example, an affinity resin that binds to LOX and removes itfrom the protein sample. The affinity resin can be linoleic acid,linolenic acid, stearic acid, oleic acid, propyl gallate, orepigalloccatechin gallate attached to a solid support such as a bead orresin. See, e.g., WO2013138793. In addition, depending on the proteincomponent of the food product, certain combinations of antioxidantsand/or LOX inhibitors can be used as effective agents to minimizeoff-flavor or off-odor generation especially in the presence of fats andoils. Such compounds can include, for example, one or more ofβ-carotene, α-tocopherol, caffeic acid, propyl gallate, orepigallocatechin gallate.

In some embodiments, specific flavor compounds, such as those describedin Tables 3, 8, 9, 12, 14, 16, or 17 can be isolated and purified fromthe flavor additive composition. These isolated and purified compoundscan be used as an ingredient to create flavors useful to the food andfragrance industry.

A flavor additive composition can be in the form, of but not limited to,soup or stew bases, bouillon, e.g., powder or cubes, flavor packets, orseasoning packets or shakers. Such flavor additive compositions can beused to modulate the flavor and/or aroma profile for a variety of foodproducts, and can be added to a consumable food product before, during,or after cooking of the food product.

Food Products

Food products containing one or more flavor precursors and one or moreheme-containing proteins can be used as a base for formulating a varietyof additional food products, including meat substitutes, soup bases,stew bases, snack foods, bouillon powders, bouillon cubes, flavorpackets, or frozen food products. Meat substitutes can be formulated,for example, as hot dogs, burgers, ground meat, sausages, steaks,filets, roasts, breasts, thighs, wings, meatballs, meatloaf, bacon,strips, fingers, nuggets, cutlets, or cubes.

In addition, food products described herein can be used to modulate thetaste and/or aroma profile of other food products (e.g., meat replicas,meat substitutes, tofu, mock duck or other gluten based vegetableproduct, textured vegetable protein such as textured soy protein, pork,fish, lamb, or poultry products such as chicken or turkey products) andcan be applied to the other food product before or during cooking. Usingthe food products described herein can provide a particular meaty tasteand smell, for example, the taste and smell of beef or bacon, to anon-meat product or to a poultry product.

Food products described herein can be packaged in various ways,including being sealed within individual packets or shakers, such thatthe composition can be sprinkled or spread on top of a food productbefore or during cooking.

Food products described herein can include additional ingredientsincluding food-grade oils such as canola, corn, sunflower, soybean,olive or coconut oil, seasoning agents such as edible salts (e.g.,sodium or potassium chloride) or herbs (e.g., rosemary, thyme, basil,sage, or mint), flavoring agents, proteins (e.g., soy protein isolate,wheat glutin, pea vicilin, and/or pea legumin), protein concentrates(e.g., soy protein concentrate), emulsifiers (e.g., lecithin), gellingagents (e.g., k-carrageenan or gelatin), fibers (e.g., bamboo filer orinulin), or minerals (e.g., iodine, zinc, and/or calcium).

Food products described herein also can include a natural coloring agentsuch as turmeric or beet juice, or an artificial coloring agent such asazo dyes, triphenylmethanes, xanthenes, quinines, indigoids, titaniumdioxide, red #3, red #40, blue #1, or yellow #5.

Food products described herein also can include meat shelf lifeextenders such as carbon monoxide, nitrites, sodium metabisulfite,Bombal, vitamin E, rosemary extract, green tea extract, catechins andother anti-oxidants.

Food products described herein can be free of animal products (e.g.,animal heme-containing proteins or other animal products).

In some embodiments, the food products can be soy-free, wheat-free,yeast-free, MSG-free, and/or free of protein hydrolysis products, andcan taste meaty, highly savory, and without off odors or flavors.

Assessment of Food Products

Food products described herein can be assessed using trained humanpanelists. The evaluations can involve eyeing, feeling, chewing, andtasting of the product to judge product appearance, color, integrity,texture, flavor, and mouth feel, etc. Panelists can be served samplesunder red or under white light. Samples can be assigned randomthree-digit numbers and rotated in ballot position to prevent bias.Sensory judgments can be scaled for “acceptance” or “likeability” or usespecial terminology. For example, letter scales (A for excellent, B forgood, C for poor) or number scales may be used (1=dislike, 2=fair,3=good; 4=very good; 5=excellent). A scale can be used to rate theoverall acceptability or quality of the food product or specific qualityattributes such beefiness, texture, and flavor. Panelists can beencouraged to rinse their mouths with water between samples, and givenopportunity to comment on each sample.

In some embodiments, a food product described herein can be compared toanother food product (e.g., meat or meat substitute) based uponolfactometer readings. In various embodiments, the olfactometer can beused to assess odor concentration and odor thresholds, odorsuprathresholds with comparison to a reference gas, hedonic scale scoresto determine the degree of appreciation, or relative intensity of odors.

In some embodiments, an olfactometer allows the training and automaticevaluation of expert panels. In some embodiments, a food productdescribed herein causes similar or identical olfactometer readings. Insome embodiments, the differences between flavors generated using themethods of the invention and meat are sufficiently small to be below thedetection threshold of human perception.

In some embodiments, volatile chemicals identified using GCMS can beevaluated. For example, a human can rate the experience of smelling thechemical responsible for a certain peak. This information could be usedto further refine the profile of flavor and aroma compounds producedusing a heme-containing protein and one or more flavor precursors.

Characteristic flavor and fragrance components are mostly producedduring the cooking process by chemical reactions molecules includingamino acids, fats and sugars which are found in plants as well as meat.Therefore, in some embodiments, a food product is tested for similarityto meat during or after cooking. In some embodiments human ratings,human evaluation, olfactometer readings, or GCMS measurements, orcombinations thereof, are used to create an olfactory map of the foodproduct. Similarly, an olfactory map of the food product, for example, ameat replica, can be created. These maps can be compared to assess howsimilar the cooked food product is to meat.

In some embodiments, the olfactory map of the food product during orafter cooking is similar to or indistinguishable from that of cooked orcooking meat. In some embodiments the similarity is sufficient to bebeyond the detection threshold of human perception. The food product canbe created so its characteristics are similar to a food product aftercooking, but the uncooked food product may have properties that aredifferent from the predicate food product prior to cooking.

These results will demonstrate that the compositions of the inventionare judged as acceptably equivalent to real meat products. Additionally,these results can demonstrate that compositions of the invention arepreferred by panelist over other commercially available meatsubstitutes. So, in some embodiments the present invention provides forconsumables that are significantly similar to traditional meats and aremore meat like than previously known meat alternatives.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1: Addition of Heme-Protein Increases Beefy Qualitiesof Replica Burgers

Replica burgers containing the ingredients in Table 4 and the flavorprecursors cysteine (10 mM), glutamic acid (10 mM), glucose (10 mM), andthiamine (1 mM) were prepared. Water was added to make up the balance.See, for example, U.S. Provisional Application No. 61/751,816, filedJan. 11, 2013. Control burgers were prepared as in Table 4 withprecursors cysteine (10 mM), glutamic acid (10 mM), glucose (10 mM), andthiamine (1 mM) except LegH was omitted.

After cooking for 5 minutes at 150 C, the replica burgers were evaluatedby a trained sensory panel. Panelists were served samples under redlights and each panelist individually evaluated the samples. Sampleswere assigned a random three-digit number and rotated in ballot positionto prevent bias. Panelists were asked to evaluate cooked replica burgersamples on multiple flavor, aroma, taste, texture and appearanceattributes including but not limited to: beefiness, bloody quality,savory quality, and overall acceptability using a 7-point scale from1=dislike extremely, to 7=like extremely. Panelists were encouraged torinse their mouths with water between samples, and to fill out a surveyto record their evaluation of each sample.

When replica burgers containing the LegH were compared to the controlreplica burgers without LegH, the samples containing LegH were ratedsignificantly beefier, bloodier, more savory, and overall preferredcompared to those that did not include LegH. See Table 5.

TABLE 4 Replica Burger Ingredients Replica burger % precooked w/w Peavicilin 3.86 Soy protein concentrate (SPC) 2.52 Bamboo fiber 0.34 NaCl0.54 Pea legumin 2 Soy Protein Isolate (SPI) (Solae, St. Louis, 4.68 MO)Wheat gluten 4.68 Coconut oil 15 Soy lecithin 0.1 k-carrageenan 1 LegH 1

TABLE 5 Sensory evaluation of replica burger with Heme Beef No 1%Attribute 20/80 Heme Heme Beefyness mean 5.33 1.30 3.20 STDEV 1.58 0.670.79 Bloody mean 4.00 1.10 2.78 STDEV 1.32 0.32 1.64 Savory mean 4.673.00 5.10 STDEV 1.22 1.63 0.57

Example 2: Replica Burgers with a Flavor Precursor Mixture Taste Beefyand Bloody

Replica burgers containing a flavor precursor mixture of glucose,cysteine, thiamine, and glutamic acid and 1% LegH pre-cooked w/w (seeTable 4) were prepared as described in Example 1, and evaluated by atrained sensory panel after the burgers were cooked for 5 minutes at 150C. Control burgers included LegH and all other ingredients except forthe flavor precursor mixture.

Panelists were asked to evaluate the samples overall improvement intaste and descriptively analyze each sample using a 5-point scale from1=dislike extremely, to 5=like extremely. Panelists were encouraged torinse their mouths with water between samples, and to fill out a surveyto record their evaluation of each sample. The replicate burgers whichincluded LegH and the flavor precursor mixture were described as havingbouillon, gravy, meaty, bloody, savory, and beefy notes on taste, andwere preferred to the same replica burger with LegH but no added flavorprecursor mixture. See, Table 6

TABLE 6 Improvement of overall taste with precursors added to LegHburgers with precursors without precursors Average 3.5 1.8 STDV 0.6 0.5

Example 3: Replica Burgers with Flavor Precursor Mixture Resulting in aBacon Taste

Replica burgers (see Table 4) were cooked with different precursor mixes(see Table 7) and 1% LegH and evaluated by a trained sensory panel afterthe burgers were cooked for 5 minutes at 150 C. Control burgerscontained LegH and all of the other ingredients except for the flavorprecursors. Panelists were asked to evaluate each sample anddescriptively analyze of each sample. 5-point scale from 1=dislikeextremely, to 5=like extremely. Panelists were encouraged to rinse theirmouths with water between samples, and to fill out a survey to recordtheir evaluation of each sample. A replica burger with a precursormixture of 10 mM glucose, 10 mM ribose, 10 mM cysteine, 1 mM thiamine, 1mM glutamic acid, 1 mM GMP, and LegH was described as having a baconaroma and taste, and overall meatiness, savory quality, a very umamiquality, a brothy quality, and slight beefy notes. See Table 7 for asummary of the flavor description for the various combinations of flavorprecursors and heme-containing protein.

TABLE 7 Flavors generated by addition of precursors to LegH (1%)Precursor (concentration) Flavor Description ribose cysteine some kindof cold-cut/sliced deli meat (10 mM) (10 mM) ribose cysteine IMP breadcrust with beef drippings, sweet, grassy, umami (10 mM) (10 mM) (2 mM)ribose cysteine lactic acid bready, malty, browned, breadcrust (10 mM)(10 mM) (1 mM) ribose cysteine lysine savory, beefy, little grassy,brothy, bread (10 mM) (10 mM) (5 mM) ribose cysteine alanine savory,weak beefy, brothy, little metallic (10 mM) (10 mM) (5 mM) ribosecysteine I + G savory, weak beefy, brothy, sweet (10 mM) (10 mM) (2 mM)ribose cysteine methionine cooked potato (10 mM) (10 mM) ribose cysteineglutamic acid little meaty, pretzel, brothy, savory, sweet, chocolate(10 mM) (10 mM) (5 mM) glucose ribose cysteine thiamine glutamic acidslight beefy, browned, grasssy, (10 mM) (10 mM) (10 mM) (2 mM) (5 mM)glucose ribose cysteine thiamine glutamic acid IMP bacon, very umami,savory, brothy, slight beef (10 mM) (10 mM) (10 mM) (2 mM) (5 mM) (2 mM)glucose cysteine thiamine glutamic acid beef jerky, bloody, meaty,brothy (10 mM) (10 mM) (2 mM) (5 mM) glucose cysteine thiamine glutamicacid lactic acid savory, beefy, bloody, meaty, savory, gravy (10 mM) (10mM) (2 mM) (5 mM) (1 mM) glucose cysteine thiamine glutamic acid lysineroast beef (10 mM) (10 mM) (2 mM) (5 mM) (5 mM) glucose cysteinethiamine glutamic acid alanine boiled beef, sweet (10 mM) (10 mM) (2 mM)(5 mM) (5 mM) glucose cysteine thiamine glutamic acid I + G beefy with asulfury note (10 mM) (10 mM) (2 mM) (5 mM) (2 mM) glucose cysteine I + Gsweet, malty, umami, meaty (10 mM) (10 mM) (2 mM) glucose I + G savory,roast beef, grassy (10 mM) (2 mM) glucose glutamic acid umami, savory,meaty, sweaty, fermented (10 mM) (5 mM)

Example 4: Type of Sugar Modulates Flavor Compounds Created in thePresence of Hemeprotein

The addition of different sugars to flavor reaction mixtures containinga hemeprotein and one or more flavor precursor compounds resulted indistinct differences in the flavor compounds generated and the overallflavor profile. LegH heme protein at 1% pre-cooked w/w/ was mixed withcysteine (10 mM) and glucose (20 mM) at pH 6 in phosphate buffer to forma flavor reaction mixture and heated to 150 C for 3 minutes; thisreaction created flavor compounds known to be present in meat; see Table8. Similarly, a flavor reaction mixture made when LegH heme protein at1% was mixed with cysteine (10 mM) and ribose (20 mM) at pH 6 and heatedto 150 C for 3 minutes created flavor compounds known to be in meat; seeTable 8.

The characteristic flavor and fragrance components were mostly producedduring the cooking process when the flavor precursor molecules reactedwith the heme-protein. Gas chromatography-mass spectrometry (GCMS) is amethod that combines the features of gas-liquid chromatography and massspectrometry to separate and identify different substances within a testsample. Samples were evaluated by GCMS to identify the flavor compoundsgenerated after heating and also evaluated for their sensory profiles.Volatile chemicals were isolated from the head space around the flavorreactions. The profile of the volatile chemicals in the headspace aroundthe flavor reaction mixtures is shown in Table 8. In particular, the useof ribose created some additional compounds as compared to glucose, asshown in Table 8.

Notably, the control mixtures of cysteine with ribose or glucose heatedin the absence of the LegH heme-protein did not generate the same set offlavor compounds. The flavor reaction mixtures containing LegH also wereevaluated by a blinded trained sensory panel, which described thesamples with ribose as having beefy, savory, brothy, and gravy-likenotes, and the samples with glucose as savory, bloody, metallic, rawmeat, and bouillon-like.

TABLE 8 Flavor compounds generated with cysteine, LegH, and eitherglucose or ribose in the flavor reaction mixture. LegH 1% cysteinecysteine (10 mM), (10 mM), Compounds glucose ribose created (20 mM) (20mM) benzaldehyde X X 2-butanone X X dimethyl trisulfide X X2-pentyl-furan X X 2-methyl-propanal X X thiazole X X butyrolactone X X2-acetylthiazole X X pentanal X X 3-methyl-butanal X X methyl-thiirane XX nonanal X X heptanal X X 2,3-butanedione X X 1,3,5-cycloheptatriene XX propyl-cyclopropane X X 2-hexyl-furan X X butanal X X 2-methyl-butanalX X 2-ethyl-furan X 2-octanone X X propanal X X trichloromethane X2-methyl-furan X X furan X X pyrazine X X thiophene X X1,3-dimethyl-benzene X X octane X octanal X X thiazole X X 2-pentanone Xfurfural X X 2-nonanone X X (Z)-2-heptenal X X (E)-2-heptenal X X1-octene X X formic acid, heptyl ester X X 2-pentyl-thiophene X1-octen-3-one X X 3-pentyl-furan X X 2-propenal X (E)-2-tridecen-1-ol Xbenzene X (E)-4-octene X 1-penten-3-one X 4-penten-2-one X X2-methyl-thiazole X methyl-pyrazine X trans-2-(2-pentenyl)furan X3-ethylcyclopentanone X pyrrole X X 2-thiophenecarboxaldehyde X3-thiophenecarboxaldehyde X

Example 5: Heme-Protein in the Presence of Thiamine Affects theProduction of Certain Flavor Compounds

The addition of thiamine in a flavor reaction mixtures with a hemeprotein and other flavor precursors affected the formation of5-Thiazoleethanol, 4-methyl-furan, 3,3′-dithiobis[2-methyl-thiazole, and4-methylthiazole. These compounds are known to be present in meat andhave beefy, meaty taste notes.

Flavor reaction mixtures at pH 6 containing LegH (1%), cysteine (10 mM),thiamine (1 mM), either glucose or ribose (20 mM), and with or withoutglutamic acid (10 mM) were prepared and subsequently heated to 150 C for3 minutes. These flavor reaction samples then were evaluated by GCMS forthe flavor compounds generated and evaluated by a trained panel fortheir sensory profiles. Volatile chemicals were isolated from the headspace around the flavor reactions. GCMS showed4-methyl-5-thiazoleethanol, 3,3′-dithiobis[2-methyl]-furan, and4-methylthiazole compounds were created by a mixture of LegH withthiamine, a sugar (either glucose or ribose), and cysteine. The sameflavor reaction mixtures without thiamine did not generate thesecompounds; additionally these compounds were not generated whenheme-proteins were not present in the flavor reaction mixtures.

The flavor reaction samples also were evaluated by a blinded trainedsensory panel, which described the samples with the addition of thiamineas more complex in taste and more beefy, meaty, and savory.

Example 6: Heme-Proteins with Nucleotides Controls Particular FlavorCompound Production

The addition of inosine monophosphate and guanosine monophosphate inmixes with heme protein and other precursors controlled the formation offlavor compounds (E)-4-octene, 2-ethyl-furan, 2-pentanone,2,3-butanedione, 2-methyl-thiazole, methyl-pyrazine, tridecane,(E)-2-octenal, 2-thiophenecarboxaldehyde, and 3-thiophenecarboxaldehyde.These compounds are known to be present in meat and have a beefy, meaty,buttery, and or savory flavor notes.

Reactions containing heme protein at 1% (LegH) with cysteine (10 mM),and glucose (20 mM), 1 mM IMP and 1 mM GMP, at pH 6.0 were prepared andheated to 150 C for 3 minutes. Characteristic flavor and fragrancecomponents were mostly produced during the cooking process whereprecursors reacting heme-protein. These samples were evaluated by GCMSfor the flavor compounds generated and evaluated for the sensoryexperience. Volatile chemicals were isolated from the head space aroundthe flavor reaction and identified using GCMS, creating a profile of thevolatile chemicals in the headspace around the flavor reaction mixture.GCMS showed 4-octene, 2-ethyl furan, 2-pentanone, 2,3-butanedione,2-methyl-thiazole, methyl-pyrazine, tridecane, 2-octenal,2-thiophenecarboxaldehyde, 3-thiophenecarboxaldehyde compounds werecreated by a mixture of hemeprotein LegH with IMP, GMP, glucose, andcysteine. The same samples without IMP and GMP did not generate thesecompounds, additionally these compounds were also not created whenheme-proteins were not present, just precursor molecules. Sensoryevaluation by blinded trained panelist found the samples with theaddition of inosine and guanosine as described as having more complexityin taste and more beefy, meaty, brothy and savory. FIG. 2 shows theabundance of the novel flavor compounds created with heme protein at 1%was mixed in a reaction at pH 6, with cysteine (10 mM), and glucose (20mM), IMP (1 mM) and GMP (1 mM), and detected by solid phasemicroextraction (SPME) and then detected by GCMS.

Example 7: Flavor Generation with the Addition of a Particular OrganicAcid

The addition of lactic acid in mixes with heme protein, ribose, andcysteine controlled the formation of the flavor compounds nonanal,thiazole, 2-acetylthiazole, and 8-methyl-1-undecene. These compounds areknown to be present in meat.

Reactions containing heme protein at 1%, cysteine (10 mM), and ribose(20 mM), and lactic acid (1 mM), pH 6.0, were prepared and heated to 150C for 3 minutes. Characteristic flavor and fragrance components weremostly produced during the cooking process where precursors reactingheme-protein. These samples were evaluated by GCMS for the flavorcompounds generated and evaluated for the sensory experience. Volatilechemicals were isolated from the head space around the flavor reactionand identified using GCMS, creating a profile of the generatedcompounds. Nonanal, thiazole, 2-acetylthiazole, and 8-methyl-1-undecenecompounds were created by a mixture of LegH with lactic acid, ribose,and cysteine. The same samples without lactic acid did not generatethese compounds, additionally these compounds were not created in theabsence of heme-proteins.

Sensory evaluation by blinded trained panelist found the samples withthe addition of lactic acid as described as beefy, savory, browned,bready, and having malty notes. The sample with everything but lacticacid rated lower in browned, bready and malty notes.

Example 8: Flavor Generated with the Addition of a Particular Amino Acid

The addition of lysine in mixes with heme protein ribose, and cysteinecontrolled the formation of flavor compounds dimethyl trisulfide,nonanal, 2-pentyl-thiophene, furfural, 2-nonenal, 1-octanol, 2-nonenal,thiazole, 2-acetylthiazole, phenylacetaldehyde, 2-acetylthiazole. Thesecompounds are known to be present in meat and some have a beefy, meaty,and or savory flavor.

Reactions containing heme protein at 1%, cysteine (10 mM), and ribose(20 mM), and lysine (1 mM), at pH 6.0, were prepared and heated to 150 Cfor 3 minutes. These samples were evaluated by GCMS for the flavorcompounds generated and evaluated for the sensory experience.Characteristic flavor and fragrance components were mostly producedduring the cooking process where precursors could react with theheme-protein. These samples were evaluated by GCMS for the flavorcompounds generated and evaluated for the sensory experience. Volatilechemicals were isolated from the head space around the flavor reaction.Dimethyl trisulfide, nonanal, 2-pentyl-thiophene, furfural, 2-nonenal,1-octanol, 2-nonenal, thiazole, 2-acetylthiazole, phenylacetaldehyde,2-acetylthiazole compounds were created by a mixture of LegH with lacticacid, ribose, and cysteine. The same samples without lactic acid did notgenerate these compounds, additionally these compounds were not createdwhen heme-proteins were not present, just precursor molecules. Sensoryevaluation by blinded trained panelist found the samples with theaddition of lysine as described as roast beefy, savory, and browned. Theaddition of lysine increased the roasted browned notes.

Example 9—Flavor Compound Production by Different Heme-Proteins

The addition of different types of heme-proteins (LegH, Barley, B.myoglobin, or A. aeolicus) in flavor reaction mixtures containing one ormore flavor precursor compounds results in many of the same key meatflavors, including but not limited to 2-pentyl-furan, 2,3-Butanedione,Thiophene, 2-methyl-thiazole, Pyrazine, Furan, Pyrrole, 2-methyl-furanand distinct differences in the flavor compounds, including but notlimited to 2-pentyl-thiophene, Nonanal, 2-Nonanone, and 1-Octen-3-one.These differences in flavor compounds can change the overall tasteprofile. The different types of heme-protein were LegH, Barley, B.myoglobin, or A. aeolicus used at 1% w/w in a reaction mixed withcysteine (10 mM) and ribose (10 mM) at pH 6. The pre-reaction mixturewas heated to 150 C for 3 minutes; this reaction created flavorcompounds known to be present in meat; see Table 9. The characteristicflavor and fragrance components are mostly produced during the cookingprocess where the flavor precursor molecules react with theheme-protein. Samples were evaluated by GCMS to identify the flavorcompounds generated after heating and also evaluated for their sensoryprofiles. Volatile chemicals were isolated from the head space aroundthe flavor reactions. Table 9 shows the similarity and differences involatile flavor compounds created by the different types ofheme-proteins.

TABLE 9 Flavor compounds created by different heme-protein when heatedwith ribose and cysteine. Name LegH Barley B. myoglobin A. aeolicusFuran x x x x Thiazole x x x x benzaldehyde x x x x 2-acetylthiazole x xx x 2-methyl-propanal x x x x furfural x x x x 2,3-butanedione x x x x2-pentyl-furan x x x x 2-pentanone x x pyrazine x x x x dimethyltrisulfide x x x x 3-methyl-butanal x x x 2-methyl-thiazole x x x xpentanal x x x x 1,3,5-cycloheptatriene x x x x methacrolein x x x xheptanal x x x x 2-methyl-butanal x x x isothiazole x x x x thiophene xx x x propanal x x x x 2-heptenal x x x methyl-pyrazine x x x x 1-octenex x x butanal x x x x 2-acetyl-propen-2-ol x x x x pyrrole x x x x2-methyl-furan x x x x nonanal x x x 2-propenal x x x 2-decenal x x x2-nonanone x x 2-octanone x x x 2-tridecen-1-ol x x 2-octanone x2-octenal x x 4-methyl-2-heptanone x x octanal x x 2-undecenal xbutyrolactone x 1-octen-3-one x 3-methylheptyl acetate x2-pentyl-thiophene x

Example 10—Generation of Meat Flavors from Different Lipids

Several different samples including oils (canola oil or coconut oil),free fatty acids (FFA) (linoleic acid (C18:2), oleic acid (C18:1),stearic acid (C18:0), or myristic acid (C14:0)) and phospholipids (PL)(beef heart polar lipids extract, Biolipon95 (from Perimond), orNatCholinePC40 (from Perimond)) were tested for their ability to producebeefy flavor in the absence and in the presents of other precursors.Oils, FFAs, and PLs were added to 50 mM potassium phosphate buffer (PPB)pH 6.0 or a Maillard reaction mix (MRM) containing 50 mM potassiumphosphate pH 6.0, 5 mM Cysteine, 10 mM Glucose, 0.1 mM Thiamine, and0.1% (w/v) LegHemoglobin. Lipids in combination with MRM were designedto capture the cross reactions of lipid degradation and Maillardreaction productions while lipids in phosphate buffer functioned as alipid control. The oils were added at 3% of the total 1 mL volume ofsolution while FFAs and PLs were added at 1% of the total 1 mL volumes.All samples were cooked at 150° C. for 3 mins, cooled to 50° C. and thenanalyzed using GCMS (SPME fiber sampling of headspace). After allsamples were analyzed by GCMS the caps were removed and samples weresmelled by a trained flavor scientist and aromas recorded.

TABLE 10 Legend showing components of each sample Sample Name SolutionAdditives MRM None Maillard Reaction Mix None MRM_Linoelic Acid MaillardReaction Mix 1% linoleic acid MRM_Oleic Acid Maillard Reaction Mix 1%oleic acid MRM_C14 Maillard Reaction Mix 1% C14:0 free fatty acidMRM_C18 Maillard Reaction Mix 1% C18:0 free fatty acid MRM_CanolaMaillard Reaction Mix 3% Canola Oil MRM_Coconut Maillard Reaction Mix 3%Coconut Oil MRM_BeefHeart Maillard Reaction Mix 1% Beef Heart PolarLipids Extract MRM_Biolipon95 Maillard Reaction Mix 1% Biolipon95(emulsifier) MRM_ Maillard Reaction Mix 1% NatCholinePC40 NatCholinePC40(emulsifier) KPhos6_Linoelic Acid PPB, pH 6 1% linoelic acidKPhos6_Oleic Acid PPB, pH 6 1% oleic acid KPhos6_C14 PPB, pH 6 1% C14:0free fatty acid KPhos6_C18 PPB, pH 6 1% C18:0 free fatty acidKPhos6_Canola PPB pH 6 3% Canola Oil KPhos6_Coconut PPB, pH 6 3% CoconutOil KPhos6_BeefHeart PPB, pH 6 1% Beef Heart Polar Lipids ExtractKPhos6_Biolipon95 PPB, pH 6 1% Biolipon95 (emulsifier) KPhos6_ PPB, pH 61% NatCholinePC40 NatCholinePC40 (emulsifier)

Table 11 contains the aroma descriptions and Table 12 contains the GCMSdata from the most interesting samples analyzed. Many of the lipidsintroduced a “fatty” aroma to MRM that was otherwise absent. Thecombinations of Linoleic Acid or NatCholinePC40 in MRM produced thegreatest abundance of fatty compounds suggesting that these lipids mayimprove the flavor perception of beef tallow. Linoleic Acid andNatCholinePC40 also showed high abundance of earthy-mushroom aromas. Theaddition of lipids to MRM significantly increased the abundance of“nutty & roasted” aromas. Less desirable “green” aroma compounds weremost prominent in samples with unsaturated free fatty acids (linoleicacid or oleic acid) or phospholipids. In general, the addition of lipidssignificantly increased the number of target beef compounds made.

TABLE 11 Aroma descriptions of each sample after it was cooked. SampleNames Aroma Descriptions MRM_Only brothy, malty, beef stewKPhos6_BeefHeart fatty, creamy, beef tallow, slight sweet, slightroasted nutty MRM_BeefHeart fatty, beef tallow, old meat, mushroomKPhos6_Biolipon95 fatty, fresh MRM_Biolipon95 fatty, brothy, hay, maltygreen KPhos6_NatCholinePC40 light fatty, fresh MRM_NatCholinePC40 fatty,beef tallow, brothy K-Phos6_C14 light/faint plastic/waxy MRM_C14 brothy,beefy, minty, fresh K-Phos6_C18 light/faint plastic/waxy MRM_C18 beefywith cucumber &/or pepper aroma K-Phos6_Canola fresh, cucumberMRM_Canola fatty, brothy, oil, roasted nuts K-Phos6_Coconut nothingMRM_Coconut brothy, beefy, slight fatty, crackers K-Phos6_Oleic Acidfresh, cucumber, camphorous/minty-like MRM_Oleic Acid herbal, plastic,slight cheesy, brothy K-Phos6_Linoelic Acid light plastic MRM_LinoelicAcid fatty, light waxy, brothy, herbal

TABLE 12 List of aromatic compounds found in Beef by GCMS and a chartshowing which were detected in each lipid plus MRM sample. MRM_Compounds in Beef MRM only MRM_BeefHeart NatCholinePC40 MRM_Linoleicacid (s)-isopropyl lactate N N N N 1-ethyl-5- Y Y Y Y methylcyclopentene1-heptanol N Y N N 1-hepten-3-ol N Y Y Y 1-heptene N Y Y Y2-methyl-1-heptene N N N N 1-hexanol N Y Y Y 2-ethyl-1-hexanol N N N N1-nonanol N N Y N 1-nonene N Y Y N 1-octanol N Y Y N 1-octen-3-ol N Y YY 1-octen-3-one Y Y Y Y 1-octene N N N N 1-pentanol N Y Y Y1-penten-3-ol N Y Y N 1-propanol N N N N 8-methyl-1-undecene N Y Y Y1,3-hexadiene N N N Y 3-ethyl-2-methyl-1,3- N Y Y Y hexadiene1,3-octadiene Y N N Y 1,3,5-cycloheptatriene N N N N2,3-dihydro-5,6-dimethyl- N N N N 1,4-dioxin 1,7-octadien-3-ol N Y N N1h-pyrrole-2- N N N N carboxaldehyde 2-methyl-1H-pyrrole N N N N2-acetyl-2-thiazoline Y N N N 2-acetylthiazole Y Y Y Y 2-butanone N Y YY 2-butenal N Y Y Y 2-ethyl-2-butenal N N N Y 3-methyl-2-butenal N N Y Y3-methyl-2-cyclohexen-1- N N N N one 2-decanone Y Y Y N (E)-2-decenal NN N N (Z)-2-decenal Y Y Y Y 2-furanmethanol N N N N 2-heptanone Y Y Y Y6-methyl-2-heptanone N N Y N (E)-2-heptenal N Y Y Y (Z)-2-heptenal N N NY (E)-2-hexenal N Y Y Y 2-ethyl-2-hexenal N N N N 2-methyl-2-heptene Y NN N 2-n-heptylfuran Y N N N 2-n-octylfuran Y Y Y N 2-nonanone N Y Y N(E)-2-nonenal Y Y Y Y (Z)-2-nonenal N N N Y 2-octanone Y Y Y Y(Z)-2-octen-1-ol Y Y Y Y (E)-2-octenal N Y Y Y 2-pentanone N Y Y N1-propoxy-2-propanol N N N N 1-(acetyloxy)-2-propanone Y N N N1-hydroxy-2-propanone Y N N N 2-propenal N N N Y2-thiophenecarboxaldehyde Y Y Y Y 2-undecenal N Y Y Y 2,3-butanedione NN N Y 2,3-pentanedione N N N N (E,E)-2,4-decadienal N Y Y Y2,4-decadienal N N N Y (E,E)-2,4-heptadienal N Y Y Y(E,E)-2,4-nonadienal N Y Y Y 2,6-dimethylpyrazine N N N N(E,Z)-2,6-nonadienal N N Y N 5-ethyldihydro-2(3H)- N Y Y Y furanone5-methyl-2(3H)-furanone N N N N dihydro-5-pentyl-2(3H)- N N Y Y furanonedihydro-5-propyl-2(3H)- N N N N furanone 2(5H)-furanone N N N Ntetrahydro-6-methyl-2H- N N N N pyran-2-one 3-ethylcyclopentanone N Y YY 3-hexanone N N N N 3-methyl-2- N N N N thiophenecarboxaldehyde3-octanone Y Y N Y 3-octen-2-one N Y Y Y 3-thiophenecarboxaldehyde N Y YY (E,E)-3,5-octadien-2-one N N Y Y dihydro-2-methyl-3(2H)- N N N Nfuranone 4-cyanocyclohexene N N N N 4-cyclopentene-1,3-dione N N Y N4-decyne N Y N N (Z)-4-heptenal N Y Y Y 4-methyloctanoic acid N N N N(E)-4-octene N N N N 2,3-dihydro-3,5-dihydroxy- Y N N N6-methyl-4(H)-pyran-4-one 6-methyl-5-hepten-2-one Y N N N acetaldehyde NN N Y acetic acid N N N N acetic acid ethenyl ester Y N N N acetoin Y NN N acetone Y N N Y acetonitrile N N N Y benzaldehyde Y Y Y Y4-ethyl-benzaldehyde N Y Y N benzene Y N N N benzoic acid, hydrazide Y NN N butanal Y N N Y 2-methyl-butanal N N N N 3-methyl-butanal Y N N Nbutanoic acid N N N N butyrolactone Y Y N Y caprolactam N N N N carbondisulfide N N N Y 1-ethyl-1-methyl- Y Y Y Y cyclopentanepropyl-cyclopropane N N Y Y decanal N Y Y N dihydro-3-(2H)-thiophenone NN N N Dimethyl sulfide Y N N N dimethyl sulfone N N N N dimethyltrisulfide Y Y N N ethanethiol N N N N ethanol N N N Y1-(1(H)-pyrrol-2-yl)-ethanone N N N N 1-(2-furanyl)-ethanone N N N Nethosuximide Y N N N formic acid, heptyl ester Y Y N N furan Y N N Y2-ethyl-furan Y N N N 2-hexyl-furan Y N N Y 2-methyl-furan N N N Y2-pentyl-furan N Y Y Y 2-propyl-furan N N Y Y 3-methyl-furan Y N N N3-pentyl-furan Y Y Y Y furfural N Y Y Y heptanal N Y Y Y heptanoic acidN N N Y 2-methyl-hex-2-yn-4-one N N N N hexanoic acid N N N Y hydrogensulfide N N N N m-aminophenylacetylene N N N N maleic anhydride N N N Nmethacrolein N N N N methanethiol N N N N methyl ethanoate N N N Nmethyl isobutyl ketone Y N N N n-caproic acid vinyl ester N Y Y Nnonanal N Y Y Y 3-methyl-nonane Y N N N nonanoic acid Y N N N octanal NY Y Y octane N N N Y octanoic acid N N N Y oxalic acid, isobutyl pentylY N N N ester p-cresol N N N N pentanal N N N Y pentanoic acid Y N N Y4-ethyl-phenol N Y Y N phenylacetaldehyde Y Y Y Y (p-hydroxyphenyl)- Y NN N phosphonic acid propanal N N N Y 2-methyl-propanal N N N N propanoicacid N N N N 2-methyl-propanoic acid Y N N N propanoic acid, ethenylester N N N N pyrazine N Y N Y 2-ethyl-5-methyl-pyrazine N N N N2-ethyl-6-methyl-pyrazine N N N N 2,3-dimethyl-pyrazine N N N N2,5-dimethyl-pyrazine N N N N 3-ethyl-2,5-dimethyl-pyrazine Y N N Nethyl-pyrazine N N N N methyl-pyrazine N N N N trimethyl-pyrazine Y N NN pyridine Y N Y N pyrrole Y Y Y Y styrene Y N Y N thiazole Y Y Y Ymethyl-thiirane N N N N thiophene N N N Y 2-hexyl-thiophene Y N Y N2-pentyl-thiophene N Y N N trans-2-(2-pentenyl)furan N Y Y Ntrans-3-nonen-2-one N Y Y Y undecanoic acid N N N N Total # of Compounds54 63 66 76 Detected:

In samples having fatty or creamy aromas, 2,4-decadienal,(E,E)-2,4-nonadienal, (E,E)-2,4-heptadienal, and/or (E,E)-2,4-decadienalwere detected in the KPhos6_BeefHeart, MRM_BeefHeart, MRM_BioLipon95,MRM_NatCholinePC40, Kphos6_Canola, MRM_Canola, KPhos6_Oleic Acid,KPhos6_Linoleic acid and MRM_Linoleic acid samples. For(E,E)-2,4-decadienal, the strongest signal intensity was in theMRM_NatCholinePC40 sample, followed by the MRM_Linoleic acid,KPhos6_Linoleic acid, MRM_BeefHeart, MRM_BioLipon95, KPhos6_BeefHeart,MRM_Oleic Acid, and KPhos6_Oleic Acid samples. For(E,E)-2,4-heptadienal, the strongest signal intensity was in theMRM_NatCholinePC40 sample followed by the MRM_Canola sample.(E,E)-2,4-heptadienal also was detected in the MRM_BioLipon95,MRM_BeefHeart, and MRM_Linoleic acid samples. For (E,E)-2,4-nonadienal,the strongest signal intensity was in the MRM_Canola and MRM_Linoleicacid samples. (E,E)-2,4-nonadienal also was detected in theKphos6_Canola, MRM_NatCholinePC40, MRM_BioLipon95, MRM_BeefHeart, andKPhos6_Linoleic acid samples. For 2,4-decadienal, the strongest signalintensity was in the MRM_Linoleic acid sample. 2,4-decadienal also wasdetected in KPhos6_Linoleic acid, MRM_Canola, and KPhos6_Oleic Acidsamples.

In samples having earthy or mushroom aromas, 3-octen-2-one,1-octen-3-one, 3-octanone, and/or 1-octen-3-ol were detected in theKPhos6_BeefHeart, MRM_BeefHeart, Kphos_BioLipon95, MRM_BioLipon95,Kphos_NatCholinePC40, MRM_NatCholinePC40, MRM_Canola, KPhos6_Oleic Acid,MRM_Oleic Acid, KPhos6_Linoleic acid, and MRM_Linoleic acid samples. For1-octen-3-ol, the strongest signal intensity was in the MRM_Linoleicacid sample, followed by MRM_NatCholinePC40, KPhos6_Linoleic acid,MRM_BeefHeart, KPhos6_BeefHeart, MRM_Canola, MRM_BioLipon95,KPhos6_Oleic Acid, and MRM_Oleic Acid samples. 3-octanone was detectedin the MRM_Oleic Acid, KPhos6_Linoleic acid, and MRM_Linoleic acidsamples. For 1-octen-3-one, the strongest signal intensity was in theMRM_Linoleic acid and MRM_BeefHeart samples, followed by KPhos6_Linoleicacid, MRM_NatCholinePC40, KPhos6_BeefHeart, MRM_BioLipon95, MRM_OleicAcid, and KPhos6_Oleic Acid samples. For 3-octen-2-one, the strongestsignal intensity was in the KPhos6_Linoleic acid sample, followed byMRM_Linoleic acid, MRM_NatCholinePC40, KPhos6_BeefHeart, KPhos6_OleicAcid, MRM_Oleic Acid, MRM_BeefHeart, MRM_BioLipon95, MRM_Canola,Kphos_BioLipon95, and Kphos_NatCholinePC40. Pyrazine was detected in theMRM_Coconut, MRM_C18, MRM_C14, and MRM_BioLipon95 samples.

In samples having a nutty and roasted aroma, thiazole and2-acetylthiazole were the most abundant compounds detected, along withpyrazine, methyl pyrazine, trimethyl pyrazine, and3-ethyl-2,5-dimethylpyrazine. 2-acetylthiazole was detected in allsamples with MRM and most abundant in samples with MRM_Beefheat,MRM_biolipon95, MRM_Canola, and MRM_coconut. Thiazole was created insamples with MRM-Coconut, MRM_BeefHeat, MRM_Biolipon95, MRM_C14,MRM_C18, MRM_Canola, MRM_Oleic acid and MRM_Linoleic acid andMRM_NatCholinePC40. Pyrazine was present in the largest amount insamples with MRM-Coconut, followed by samples MRM_BeefHeat,MRM_Biolipon95, MRM_C14, MRM_C18, MRM_Canola having roughly equalamount, MRM_Oleic acid and MRM_Linoleic acid sample had even less.Methyl-pyrazine was present in MRM_Biolipon95 and MRM_Coconut.3-ethyl-2,5-dimethyl-pyrazine and trimethyl-pyrazine, were present onlywithout phospholipids in the MRM.

In samples having green, vegetable, or grass aromas, 1-heptanol,1-hepten-3-ol, 1-hexanol, (E)-2-heptenal, (Z)-2-heptenal, (E)-2-hexenal,2-pentyl-furan, and/or heptanal were detected in the KPhos6_BeefHeart,MRM_BeefHeart, Kphos_BioLipon95, MRM_BioLipon95, Kphos_NatCholinePC40,MRM_NatCholinePC40, Kphos_C14, MRM_C14, Kphos_C18, MRM_C18, MRM_Canola,MRM_Coconut, KPhos6_Oleic Acid, MRM_Oleic Acid, KPhos6_Linoleic acid,and MRM_Linoleic acid samples. For 2-pentyl-furan, the strongest signalintensity was in the KPhos6_BeefHeart sample, followed by theKPhos6_Linoleic acid, MRM_BioLipon95, MRM_Linoleic acid, MRM_BeefHeart,MRM_Oleic Acid, MRM_NatCholinePC40, MRM_Canola, KPhos6_Oleic Acid, andKphos_NatCholinePC40 samples. For (E)-2-heptenal, the strongest signalintensity was in the MRM_BeefHeart, MRM_Canola, MRM_Oleic Acid, andKPhos6_Linoleic acid samples, followed by the KPhos6_Oleic Acid,MRM_BioLipon95, KPhos6_BeefHeart, MRM_Linoleic acid, MRM_NatCholinePC40,Kphos_BioLipon95, and Kphos_NatCholinePC40 samples. For (Z)-2-heptenal,the strongest signal intensity was in the MRM_Linoleic acid sample.MRM_Linoleic acid also was detected in the KPhos6_Linoleic acid sample.For heptanal, the strongest signal intensity was in the MRM_Oleic Acidsample, followed by the KPhos6_Oleic Acid, MRM_C14, MRM_C18, MRM_Canola,MRM_BeefHeart, MRM_NatCholinePC40, MRM_Linoleic acid, andKPhos6_BeefHeart samples. For, (E)-2-hexenal, the strongest signalintensity was in the MRM_Linoleic acid sample, followed by theMRM_NatCholinePC40, KPhos6_Linoleic acid, and MRM_Oleic Acid samples.

Example 11—Creation of Beefy Flavors Using Complex Precursor Mixtures

A formulation was prepared (the “magic mix,” see Table 13 containing theestimated concentrations of amino acids, sugars, and other smallmolecules in beef based on their values reported in literature. Themagic mix was tested for its ability to produce beefy flavors in thepresence of LegHemoglobin (LegH). The magic mix and 1% w/v LegH wereadded to the meat replica, pH 6.0 (see Table 4) and baked in aconvection oven for 7 minutes at 160° C. A control sample was preparedby adding 1% w/v LegH to the meat replica, pH 6.0 and baking in aconvection oven for 7 minutes at 160° C.

The meat replica sample containing only LegH, was compared to the meatreplica sample containing the magic mix and LegH by a sensory panel andGCMS analysis. Five tasters rated the flavored meat replicas forbeefiness, bitterness, and levels of savory flavors, and off flavors.Each property was rated on a 7 point scale in which 7 was the highestamount of the specified property (e.g., a standard 80:20 ground beefwould be rated 7 on the beefy scale). The Magic Mix flavor was rated onepoint higher in beefy character than the LegH only sample (FIG. 1).

To determine which chemical products were produced upon heating, asolution of Magic Mix was prepared with 1% w/v LegH at pH 6.0. Thesamples were cooked with shaking at 150° C. for three minutes, thenSolid Phase Micro Extraction (SPME) was performed for twelve minutes at50° C. to extract the volatile compounds above the headspace of thereaction. A search algorithm was used to analyze the retention time andmass fingerprint information of the volatile compounds and assignchemical names to peaks. Table 14 shows the compounds identified in boththe Magic Mix+LegH (MM, average of two samples) and in the LegH alone inbuffer (LegH, average of five samples) samples. The compounds in Table14 are listed in order of the retention time (R.T., in seconds), and aredesignated as having a zero peak area (0), or a small (S), medium (M),or large (L) average peak area. Hundreds of compounds were identifiedbetween the samples, many of which are characteristic of beefy aroma,including but not limited to 1,3-bis(1,1-dimethylethyl)-benzene,2-methyl 3-furanthiol, and Bis(2-methyl-4,5-dihydro-3-furyl) disulfide,which increased in the samples containing the Magic Mix and LegH.

TABLE 13 Chemical entities added to the Magic Mix Chemical entity mMAlanine 5.6 Arginine 0.6 Asparagine 0.8 Aspartate 0.8 Cysteine 0.8Glutamic acid 3.4 Glutamine 0.7 Glycine 1.3 Histidine 0.6 Isoleucine 0.8Leucine 0.8 Lysine 0.7 Methionine 0.7 Phenylalanine 0.6 Proline 0.9Threonine 0.8 Tryptophan 0.5 Tyrosine 0.6 Valine 0.9 glucose 5.6 Ribose6.7 Maltodextrin 5.0 Thiamine 0.5 GMP 0.24 IMP 0.6 Lactic acid 1.0creatine 1.0 NaCl 10 KCl 10 Kphos pH 6.0 10

TABLE 14 Compounds identified with GC-MS analysis in samples with MM andLegH, or LegH alone (average of five samples) MM with LegH R.T.(s) NameLegH alone 248 acetaldehyde L S 256.3 carbon disulfide L S 264.3dimethyl sulfide S 0 265 oxalic acid, isobutyl pentyl ester M 0 268.12,3,4-trimethyl-pentane M 0 269.2 methanethiol S 0 283.4 propanal M 0285.4 octane M 0 287.1 furan M 0 295.3 2-methyl-propanal L S 297.6acetone L S 319.3 2-propenal M S 338.1 2-methyl-furan M S 342.1 butanalL S 344.2 2,4-dimethyl-1-heptene M 0 346.3 methacrolein M 0 357.4methyl-thiirane L 0 360.2 3-methyl-furan S 0 363.7 butanone L S 368.92,3-dihydro-5-methyl-furan M S 376.4 2-methyl-butanal L M 381.13-methyl-butanal L M 390.6 isopropyl alcohol 0 S 399.6 ethanol L M 406.22-propenoic acid, methyl ester M 0 408.2 benzene S 0 414.4 methyl vinylketone M 0 416.4 2,2,4,6,6-pentamethyl-heptane M 0 422.6 2-ethyl-furan S0 438.4 2-ethylacrolein M 0 449.9 2-pentanone S 0 453.2pentanal/2,3-butanedione L 0 453.8 2,3-butanedione L M 472.84,7-dimethyl-undecane M S 485.9 2-methyl-pentanal M 0 492.62-methyl-1-penten-1-one S 0 496.6 (E)-3-penten-2-one M 0 508.61-penten-3-one M 0 510.6 trichloromethane M M 520.4 p-dithiane-2,5-diolM 0 525.5 3-methyl-pentanal M 0 535.1 (E)-5-decene M 0 536.5 toluene M S537.9 2-butenal M S 543.8 4-penten-2-one M 0 550.8 methyl thiolacetate M0 683.7 p-xylene S 0 727.4 dimethyl selenone M 0 738.3 methyl isopropyldisulphide M 0 755 2-heptanone M 0 758.7 heptanal L 0 781.91,3-diisopropoxy-1,3-dimethyl-1,3- S M disilacyclobutane 789.43-methyl-2-butenal M 0 793.4 4-methyl-2-heptanone M 0 810.4 pyrazine M 0818.8 isothiazole S 0 827.1 acetyl valeryl M 0 831.8 2-pentyl-furan L 0851 2-methyl-thiazole S 0 853.3 isothiocyanato-methane S 0 870.9thiazole L 0 879.2 styrene M 0 890.7 1-(methylthio)-propane M 0 895.6methyl-pyrazine M 0 910.5 thiocyanic acid, methyl ester S 0 918.64-methylthiazole M 0 921.4 2-octanone M 0 923.9 2-methyl-cyclopentanoneM 0 927.9 octanal L S 934.3 tridecane M 0 948.8trans-2-(2-pentenyl)furan S 0 961.9 1-hydroxy-2-propanone M 0 974.5(E)-2-heptenal M 0 987.4 5-methyl-1-undecene M 0 993.8 2-hexyl-furan M 01007.8 7-methyl-(E)-5-undecene M 0 1024.1 2-methyl-5-(methylthio)-furan,S 0 1058.6 2-butyl-1-decene M 0 1079.3 dimethyl trisulfide L S 1085.32-nonanone M 0 1093.2 nonanal L M 1142.31,3-bis(1,1-dimethylethyl)-benzene M 0 1149.6 (E)-2-octenal M 0 1164.51-heptanol M 0 1193.5 methional L 0 1198.8 acetic acid M S 1207.2furfural M 0 1242.1 2-decanone M 0 1250.8 decanal M 0 1265.21-decen-3-one M 0 1283.3 pyrrole M 0 1292.6 5-ethenyl-4-methyl-thiazoleM 0 1294.3 benzaldehyde L M 1303.7 2-n-octylfuran M 0 1305.6(E)-2-nonenal M 0 1341.4 1-octanol M 0 1361.1 2-methyl-1(H)-pyrrole S 01391.7 2-undecanone M 0 1401.2 (E)-2-octen-1-ol M 0 1448 butyrolactone SS 1456.3 (E)-2-decenal M 0 1462.4 phenylacetaldehyde L S 1466.32-acetylthiazole L 0 1471.3 acetophenone M S 1475.4 1-nonanol M 0 1487methyl (methylthio)methyl disulfide M 0 1497.15-(2-chloroethyl)-4-methylthiazole L 0 1497.51-(ethylthio)-2-(methylthio)-buta-1,3-diene L S 15123-thiophenecarboxaldehyde M 0 1518.8 2-nonen-4-one M 0 1531.72-thiophenecarboxaldehyde S 0 1543.9 dodecanal M 0 1551.64-ethyl-2-methyl-pyrrole S 0 1558.2 3-(methylthio)-propanenitrile S 01561.2 3-decen-2-one M 0 1613.1 bis(2-methyl-4,5-dihydro-3-furyl)disulfide M 0 1615.6 1,10-undecadiene M 0 1619.5 2-undecenal S 0 1668.92-phenylpropenal M 0 1692.3 (Z)-3-decen-1-ol, acetate M 0 1733.13-phenyl-furan S 0 1739.7 4-nitrophenyl 2-thiophenecarboxylic acid S 0ester 1741.2 5-formyl-4-methylthiazole M 0 1749.7 pentanoic acid,2,2,4-trimethyl-3-hydroxy-, M 0 isobutyl ester 1765.5 benzyl alcohol S 01774.2 pentanoic acid, 2,2,4-trimethyl-3-hydroxy-, S 0 isobutyl ester1796.9 dodecanal M 0 1806.1 (1-ethyl-1-propenyl)-benzene S 0 1825.61-undecanol M S 1827.9 2-methyl-3-furanthiol M 0 1828.32-methyl-3-(methylthio) furan M 0 1836.14-chloro-2,6-bis(1,1-dimethylethyl)-phenol S 0 1844.1 phenol S S 1845.3[(methyl sulfonyl)methyl]-benzene S 0 1850.3 (e)-2-tridecen-1-ol M 01859.9 1-heptyl-1,2,3,4-tetrahydro-4-methyl- S 0 naphthalene 1863.22,4-decadienal S 0 1905.1 3,3′-dithiobis[2-methyl]-furan M 0 1906.93,5-di-tert-butylbenzoic acid S 0 1909.6 4-ethoxy-benzoic acid, ethylester S 0 1921.5 3-(phenylmethyl)-2,5-piperazinedione S 0 1944.59-octadecenal M 0 1959.7 3,5-bis(1,1-dimethylethyl)-phenol M S 1968.44-methyl-5-thiazoleethanol M S 2007.81,1′-(1,2-cyclobutanediyl)bis-cis-benzene S 0 2019.8 benzoic acid S S2026.4 4-quinolinecarboxaldehyde S 0 2027.8 m-aminophenylacetylene M 0

Example 12—Ferrous Chlorin Catalyzes Production of Meat-Like FlavorCompounds

Fresh green spinach (10 lb) was added to 500 mL water and finely groundin a Vitamix blender to yield 2 L of green suspension. Acetone (8 L) wasadded with mixing and the material was allowed to extract for 1 hour.The material was filtered through Whatman filter paper and the acetonewas removed on a rotary evaporator (Buchi). To the residual greensuspension (500 mL) was added 2 mL of 10 M HCl, causing the suspensionto turn brown. To this was added 1 g of FeCl₂.4H₂O in 10 mL H₂O. Thesolution was shaken then left at 4° C. for 16 hours. This suspension wasextracted with diethyl ether (3×50 mL) to give a bright green organicphase, the combined organics were washed with saturated sodium chloridesolution, dried over sodium sulfate, filtered and evaporated to leave ablack paste (1.1 g). The pellet was dissolved in chloroform forfractionation.

Chlorophyll and Ferrous chlorin crude fractions were stored at −20° C.Crude extracts were fractionated by reverse-phase high-pressure liquidchromatography (RP-HPLC). HPLC conditions are outlined in Table 15. Bothchlorophyll and ferrous chlorophyll were eluted from the column with apeak retention time of 7.6 minutes. Eluted material was collected from7.3-8.0 minutes. Collected fractions were pooled and stored on ice.Collected fractions were re-chromatographed and showed a single peakwith retention time 7.6 minutes. The desired fractions were pooled, then10% sunflower oil was added, methanol was removed on a rotary evaporator(Buchi).

TABLE 15 HPLC conditions for purification of chlorophyll and ferrouschlorin from crude extract. Sample: Chlorophyll or Fe-chlorin (~2 mg/mLin CHCl₃) System: Agilent 1100 with Chemstation Column: Zorbax Bonus-RP(4.6 × 250 mm, 5 uM) Mobile phase: acetonitrile, methanol, ethyl acetate(60:20:20) isocratic flow Temperature: 30° C. Flow Rate: 1.0 mL perminute Injection volume: 0.05 mL

Preparation of Flavor Reaction Containing Ferrous Chlorin orLeghemoglobin

A solution of ferrous chlorophyll was mixed with the Magic Mix (Table13) to a final concentration of 0.35% ferrous chlorin, 1% glycerol,0.005% tween-20, 5% sunflower oil, 100 mM NaCl, 20 mM phosphate at pH 6.Leghemoglobin (0.35%) at pH 6 in phosphate buffer (20 mM), 100 mM NaCl,was mixed with the Magic Mix (Table 13), 1% glycerol, and 0.005%tween-20. The flavor reaction mixtures were heated to 150° C. for 3minutes; this reaction created flavor compounds known to be present inmeat, created by hemoglobin and also created by ferrous chlorin; seeTable 16.

The characteristic flavor and fragrance components were mostly producedduring the cooking process when the flavor precursor molecules reactedwith the heme-protein or the ferrous chlorophyll. Samples were evaluatedby GCMS to identify the flavor compounds generated after heating.Volatile chemicals were isolated from the headspace around the flavorreactions. The profile of the volatile chemicals in the headspace aroundthe flavor reaction mixtures that were similar between heme-protein andferrous chlorin are shown in Table 16. Notabily, many of the compoundscreated by the ferrous chlorin are important in the flavor of meat.

TABLE 16 Flavor Compounds created by both Ferrous Chlorin and LegH withMagic Mix. 1-heptanol acetone 1-hexanol acetonitrile 1-octanolbenzaldehyde 1-octen-3-ol butanal 1-octen-3-one 2-methyl-butanal1-pentanol dimethyl trisulfide 2-acetylthiazole ethyl acetate 2-butenalfuran 3-methyl-2-butenal, 2-ethyl-furan (Z)-2-decenal 2-hexyl furan6-methyl-2-heptanone 2-pentyl-furan (E)-2-heptenal furfural(E)-2-hexenal heptanal 2-methyl-3-furanthiol aminophenylacetylene(E)-2-nonenal methacrolein (E)-2-octenal methional 2-pentanone octanal1-hydroxy-2-propanone octane 2-thiophenecarboxaldehyde oxalic acid,diallyl ester 2-undecenal 2,3-butanedione 3-methyl-3-buten-2-one2-methyl-propanal 3-thiophenecarboxaldehyde pyrazine (E)-4-octene,2,3-dimethyl-pyrazine methyl-pyrazine 2,5-dimethyl-pyrazine thiazole

Example 13—Flavor Creation by Immobilized Hemin Preparation of HeminLinked CM Sepharose.

200 mg of bovine hemin (Sigma Aldrich) was loaded into a scintillationvial. A small magnetic stir bar, 800 μL acetonitrile, 64 μL4-methylmorpholine, and 71 mg of N-hydroxysuccinimide were added in thatorder. The vial was placed in an ice bath and chilled then 118 mg ofN-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride was addedwith stirring, followed by 845 μL of Jeffamine ED900. This was stirredwhile allowing the black mixture to warm to ambient temperature.Chloroform (10 mL) was added to the mixture followed by water (4 mL). Aflashlight was used to distinguish between organic and aqueous layerssince both were black and the organic layer was pipetted off andconcentrated to a dark black oil. The oil was dissolved in a 4:1 mixtureof acetonitrile and ethanol to make an approximately 10% strengthsolution that was inky black in color.

2 mL of water swelled and equilibrated CM Sepharose was equilibrated ina BioRad minicolumn with 3 volumes of acetonitrile. The resin wasresuspended in 1 mL acetonitrile and pipetted into a scintillation vial.This was followed with 44 microliters 4-methylmorpholine, 23 mgN-hydroxysuccinimide, and 39 mg of solidN-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride. Themixture was vortexed vigorously and then shaken for three hours. To thiswhite solid was added 570 microliters of inky black 20% strength hemincoupled diamine. The black solid was vortexed and shaken for an hour.The slurry strongly resembled Turkish coffee. The mixture was pouredinto a BioRad minicolumn and filtered, washed with acetonitrile untilwhat came out no longer resembled espresso, then switched to deionizedwater, and finally 20 mM pH 9 sodium carbonate buffer. The black solidwas washed until the effluent ran clear and then resuspended in 2 mL ofbuffer for storage until use.

Flavor Reaction

The flavor reaction was created with heme protein (equinemyoglobin-Sigma) at 0.35% in a phosphate buffer (20 mM) at pH 6.0 with100 mM NaCl, this was mixed with Magic Mix (Table 13). Another flavorreaction was created with Immobilized Hemin at 0.35% in a phosphatebuffer (20 mM) at pH 6.0 with 100 mM NaCl, this was mixed with Magic Mix(Table 13). The flavor reaction mixtures were heated to 150° C. for 3minutes; this reaction created flavor compounds known to be present inmeat.

The characteristic flavor and fragrance components were mostly producedduring the cooking process when the flavor precursor molecules reactedwith the Heme-protein or the immobilized Hemin. Samples were evaluatedby GCMS to identify the flavor compounds generated after heating.Volatile chemicals were isolated from the headspace around the flavorreactions. As can be seen in Table 17, immobilized hemin catalyzedproduction of compounds similar to those whose production was catalyzedby myoglobin free in solution. Notably, the profiles of flavorcompounds, measured by GCMS, produced by cooking mixtures containing theimmobilized hemin and the heme-protein, respectively, were very similar.

TABLE 17 Flavor compounds produced by cooking mixtures containing eithermyoglobin free in solution or hemin coupled to a solid support Flavorcompound myoglobin hemin-linker-resin 2-methyl-5-(methylthio)-thiopheneLow dihydro-5-propyl-2(3H)-furanone Low octane Low pyrrole Low Lowmethanethiol Low Low 2-thiophenecarboxaldehyde Low Low methyl-pyrazineLow Low 1-hydroxy-2-propanone Low Low propanal Low Low thiophene Lowmedium pyridine Low Low 2-methyl-furan Low medium oxalic acid, butylpropyl ester Low Low pyrazine medium Low oxalic acid, diallyl estermedium medium 2-butenal medium large furfural medium medium nonanalmedium medium 2-ethyl-furan medium Low ethanol medium very largetert-butanol medium 3,3′-dithiobis[2-methyl]-furan medium mediumm-aminophenylacetylene medium medium 2,5-dihydro-3,4-dimethyl-furanmedium medium 2-acetylthiazole medium medium cyclohexane medium ethyltert-butyl ether medium carbon di sulfide medium medium thiazole mediummedium acetonitrile medium large 2-pentyl-furan medium Low3-thiophenecarboxaldehyde medium medium 2-methyl-butanal medium mediumthiazole medium large 2-methyl-3-furanthiol larege large 2-propenallarge large 3-methyl-2-butenal large medium 2-methyl-3-(methylthio)furan large large ethyl acetate large medium methacrolein large mediummethyl-thiirane large large methional large large methyl alcohol largemedium 2-butanone large Low 2,3-butanedione large medium acetone largelarge furan large medium benzaldehyde large medium methyl thiolacetatelarge medium acetaldehyde very large very large 2-methyl-propanal verylarge very large dimethyltrisulfide very large very large3-methyl-butanal very large very large propyl-cyclopropane medium(E)-2-octenal medium 2-n-propylaziridine medium thiirane medium ethylformate medium methyl vinyl ketone medium 2-propenoic acid, ethyl estermedium 1-nonanol large 1-octene large 1-heptanol large 1-dodecene largephorone very large

Example 14. The Combination of Precursors with Heme Protein DrivesFlavor Reactions

Three samples were compared: precursor mix alone, 1% heme protein alone,and precursor mix with 1% heme. The precursor mix was made of glucose(20 mM), ribose (20 mM), cysteine (10 mM), thiamine (1 mM), and glutamicacid (1 mM). Reactions were all at pH 6.0, prepared and heated to 150°C. for 3 minutes. These three samples were run in duplicate. Thesesamples were evaluated by GCMS for the flavor compounds generated.Characteristic flavor and fragrance components were mostly producedduring the cooking process where precursors could react with theheme-protein. These samples were evaluated by GCMS for the flavorcompounds generated and evaluated for the sensory experience. Volatilechemicals were isolated from the head space around the flavor reaction.The flavor compounds created in each sample is indicated in Table 18. Asshown most of the flavor molecules were created on when the precursorsare combined with the heme protein.

TABLE 18 Flavor molecules created by the combination of LegH andprecursor mix. Precursor Precursor Compound mix LegH mix + Leg H carbondisulfide medium medium high isopropyl alcohol medium medium low2-methyl-furan low low butanal low medium thiophene low low2,3-butanedione low low high furan low medium 2,4-dimethyl-1-heptenehigh high acetone high high dimethyl trisulfide medium medium2-methyl-heptane medium medium 2-pentanone medium pentanal medium medium2-pentyl-furan medium medium 2-methyl-propanal low high2-acetatyl-1-propene low low 2-methyl-butanal low medium1,3-dimethyl-benzene low low octane low low benzene low low benzaldehydevery high 2-butanone very high furfural very high thiazole high nonanalhigh thiazole high 2-acetylthiazole medium 3-methyl-butanal medium(Z)-2-heptenal medium heptanal medium methyl-thiirane medium3-ethyl-pentane medium phenylacetaldehyde medium 2-hexyl-furan medium2-nonanone medium propanal medium pyrazine medium (Z)-2-heptenal medium2-methyl-1-heptene medium 2-ethyl-furan medium octanal medium(E)-4-octene low (E)-2-octenal low 2-methyl-thiazole low 2-propenal low1-octen-3-one low 1-octene low 2-octanone low dimethyl sulfide low3-pentyl-furan low 2-n-octylfuran low 2-pentyl-thiophene low

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. (canceled)
 2. A method of imparting a color transition to aplant-based meat analog product, comprising: including in a plant-basedmeat analog product a composition comprising: a) a heme-containingprotein, wherein the heme-containing protein is a plant heme-containingprotein, an algal heme-containing protein, a fungal heme-containingprotein, a ciliate heme-containing protein, a bacterial heme-containingprotein, or a combination thereof; and b) a stabilizer, wherein theplant-based meat analog product transitions from a substantially pink orred color before cooking to a substantially brown color during cooking.3. The method of claim 2, wherein the stabilizer comprises alipoxygenase inhibitor, an antioxidant, a shelf life extender, ormixtures thereof.
 4. The method of claim 2, wherein the antioxidantcomprises one or more of vitamin C, beta-carotene, alpha-tocopherol,caffeic acid, propyl gallate, and epigallocatechin gallate.
 5. Themethod of claim 2, wherein the shelf life extender comprises one or moreof carbon monoxide, a nitrite, sodium metabisulfite, BOMBAL®, vitamin E,rosemary extract, green tea extract, and a catechin.
 6. The method ofclaim 2, wherein the composition is included in the plant-based meatanalog product such that the heme-containing protein is present in anamount of about 0.01% to about 5% (by weight) in the plant-based meatanalog product.
 7. The method of claim 2, wherein the compositioncontains no animal products that contain heme.
 8. The method of claim 2,wherein in the absence of the composition, the plant-based meat analogproduct comprises less than 0.01% of a heme-containing protein.
 9. Amethod of imparting a meat-like flavor to a plant-based meat analogproduct, comprising: including in a plant-based meat analog product acomposition comprising: a) a heme-containing protein, wherein theheme-containing protein is a plant heme-containing protein, an algalheme-containing protein, a fungal heme-containing protein, a ciliateheme-containing protein, a bacterial heme-containing protein, or acombination thereof; and b) an emulsifier, a gelling agent, a fiber, alipoxygenase inhibitor, an antioxidant, a shelf life extender, ormixtures thereof, wherein, upon cooking the plant-based meat analogproduct, the production of at least two volatile compounds which have ameat-associated aroma is increased as compared to a plant-based meatanalog product lacking the composition.
 10. The method of claim 9,wherein the antioxidant comprises vitamin C, beta-carotene,alpha-tocopherol, caffeic acid, propyl gallate, or epigallocatechingallate.
 11. The method of claim 9, wherein the shelf life extendercomprises carbon monoxide, a nitrite, sodium metabisulfite, BOMBAL®,vitamin E, rosemary extract, green tea extract, or a catechin.
 12. Themethod of claim 9, wherein the composition is included in theplant-based meat analog product such that the heme-containing protein ispresent in an amount of about 0.01% to about 5% (by weight) in theplant-based meat analog product.
 13. The method of claim 9, wherein theplant-based meat analog product contains no animal products that containheme.
 14. The method of claim 9, wherein in the absence of thecomposition, the plant-based meat analog product comprises less than0.01% of a heme-containing protein.
 15. A plant-based meat analogproduct comprising: about 0.01% to about 5% by weight of aheme-containing protein; at least about 10% by weight of a non-animalprotein; and a non-animal fat, wherein the plant-based meat analogproduct comprises no animal products that contain heme, and wherein theplant-based meat analog product transitions from a substantially pink orred color in an uncooked state to a substantially brown color duringcooking.
 16. The plant-based meat analog product of claim 15, whereinthe non-animal protein comprises textured vegetable protein.
 17. Theplant-based meat analog product of claim 15, wherein the plant-basedmeat analog product comprises an emulsifier, a gelling agent, a fiber, alipoxygenase inhibitor, an antioxidant, a shelf life extender,histidine, or mixtures of two or more thereof.
 18. The plant-based meatanalog product of claim 17, wherein the antioxidant comprises one ormore of vitamin C, beta-carotene, alpha-tocopherol, caffeic acid, propylgallate, or epigallocatechin gallate.
 19. The plant-based meat analogproduct of claim 17, wherein the shelf life extender comprises one ormore of carbon monoxide, a nitrite, sodium metabisulfite, BOMBAL®,vitamin E, rosemary extract, green tea extract, or a catechin.
 20. Theplant-based meat analog product of claim 15, wherein the heme-containingprotein is a plant heme-containing protein, an algal heme-containingprotein, a fungal heme-containing protein, a ciliate heme-containingprotein, a bacterial heme-containing protein, or a combination thereof.21. The plant-based meat analog product of claim 15, wherein thecomposition contains no animal products that contain heme.